Compositions containing protein loaded exosome and methods for preparing and delivering the same

ABSTRACT

The present invention relates to a method for the mass-production of exosome comprising a cargo protein, a vector for preparing the exosome, exosome including a cargo protein prepared by the method, and a method for loading the cargo protein to cytosol by using the exosome prepared thereby. According to the method for preparing an exosome comprising a cargo protein provided by the present invention, the exosome loaded with a cargo protein can be produced with a high yield, so that it can be used broadly in the treatment of disease using the exosome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/KR2016/004750, filedMay 4, 2016, which claims the benefit of priority from Korean PatentApplication No. 10-2015-0062604 filed May 4, 2015, Korean PatentApplication No. 10-2015-0120934 filed Aug. 27, 2015, the contents ofeach of which are incorporated herein by reference.

This application is also a continuation-in-part of PCT/KR2017/011070,filed Sep. 30, 2017, which claims benefit from Korean Patent ApplicationNo. 10-2016-0126335 filed Sep. 30, 2016, Korean Patent Application No.10-2016-0126921 filed Sep. 30, 2016, Korean Patent Application No.10-2016-0126961 filed Sep. 30, 2016, Korean Patent Application No.10-2016-0127486 filed Oct. 4, 2016, Korean Patent Application No.10-2016-0132616 filed Oct. 13, 2016, Korean Patent Application No.10-2017-0018637 filed Feb. 10, 2017, the contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions containing protein loadedexosome, methods for preparing exosome loaded with a cargo protein usinga photo-specific binding protein, and a method for delivering the cargoprotein to cytosol using the exosome prepared thereby.

BACKGROUND OF THE INVENTION

The human body is composed of about 200 kinds of 100 trillion cells, inwhich the physiological activity is regulated by the action of variousproteins to maintain life.

Cells are surrounded by membranes in bilayer structure composed ofphospholipids, which block the entry of foreign substances into cells.Most of the protein drugs which have developed so far cannot passthrough the cell membrane to enter the cell and can act on the outsideof the cell or act on a receptor on the cell membrane to deliver thesignal into the cell in order to show physiological effect.

Cytosol has lots of proteins which interact with each other to regulatephysiological activity. So, if only a protein drug can be deliveredinside the cell, that is, inside the cytosol, the cell activity would becontrolled more effectively.

Recently, studies have been actively going on to establish a method fordelivering a cargo protein directly into cells via cell membrane. When arecombinant protein of a cargo protein and protein transduction domains(PTDs), the peptide that passes through the cell membrane, is preparedand administered, it can enter the cytosol through the cell membrane(FIG. 1). PTD is exemplified by HIV-1 TAT, HSV VP22, Antp, dfTAT, andHph-1. A fusion protein prepared by combining the PTDs and a cargoprotein is produced as a recombinant protein and at this time aseparation process is required. However, this process is problematic inthat the protein refolding is not performed properly, the activity isdecreased, the protein is nonspecifically transferred, the risk ofcausing an immune reaction in vivo is large, the cost is high, and theyield is low.

The cargo protein conjugated with various nanoparticles can enter thecytosol through the cell membrane by endocytosis (FIG. 2). At this time,the nanoparticles are exemplified by Gold NP, Liposome NP, Magnetic NP,and Polymeric NP, etc. The separation of the nanoparticles from thecargo protein occurs mostly in lysosome in the cell, so the cargoprotein is decomposed inside lysosome to lose its activity. Or thenanoparticles are difficult to be separated from the cargo protein incytosol and toxicity of the nanoparticles can be another problem.

Exosome is a small vesicle with a membrane structure in the size of50˜200 nm, which is secreted out of the cell with containing protein,DNA, and RNA for intercellular signaling.

Exosome was first found in the process of leaving only hemoglobin in thered blood cells by eliminating intracellular proteins at the last stageof red blood cell maturation. From the observation under electronmicroscope, it was confirmed that exosome is not separated directly fromplasma membrane but discharged extracellular from the intracellularspecific zone, called multi-vesicular bodies (MVBs). That is, when MVBsare fused with plasma membrane, such vesicles are discharged outside ofthe cell, which are called exosome (FIG. 3).

It has not been clearly disclosed the molecular mechanism of the exosomegeneration. However, it is known that various immune cells includingB-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, andmacrophages, stem cells, and tumor cells produce and secrete exosomeswhen they are alive.

Exosome contains various intracellular proteins, DNA, and RNA. Thesubstances secreted out of the cells contained in these exosomes can bereintroduced into other cells by fusion or endocytosis and serve asintercellular messengers. By analyzing such substances that are secretedout of the cell as included in exosome, specific disease can bediagnosed.

Exosome also includes various types of microRNAs. A method fordiagnosing a disease by detecting the presence or absence and theabundance thereof has been reported (KR 10-2010-0127768A). InternationalPatent Publication No. WO2009-015357A describes a method for predictingand diagnosing a specific disease by detecting exosome in the cancerpatient originated samples (blood, saliva, tears, etc.). In particular,the exosome obtained from a patient having a specific disease (lungdisease) is analyzed and the relationship between a specific microRNAand lung disease is specifically described. Studies have been stillgoing on to establish a method to diagnose kidney disease, in additionto lung disease, by using a specific protein included in exosome.

Exosome might also include antigens. In antigen presenting cells (APC),antigen peptide is loaded in MHC (major histocompatibility complex)class II molecule in the intracellular compartment having a membranestructure including polycystic bodies. Therefore, the exosome originatedtherefrom also has the antigen peptide-MHC class II complex. So, exosomeacts as an immunogen carrier to present antigen peptide to CD4+Tlymphocytes and thereby can induce immune response such as T lymphocyteproliferation. The molecules that are able to stimulate immune responsesuch as MHC class I and heat-shock proteins (HSPs) are concentrated inexosome, so that exosome can be used to increase or decrease immuneresponse for the treatment of cancer or auto-immune disease.

SUMMARY OF THE INVENTION

The present invention provides compositions containing exosome loadedwith a cargo protein.

In another embodiment, the present invention provides a method forpreparing the exosome loaded with a cargo protein using a photo-specificbinding protein.

In a further embodiment, the present invention provides a method ofdelivering the cargo protein to cytosol using the exosome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the method for delivering a cargo protein through arecombinant protein of a cargo protein and protein transduction domains(PTDs) (Steven R. et al. Protein transduction: unrestricted deliveryinto all cells Trends in Cell Biology, 2000).

FIG. 2 illustrates the method for delivering a cargo protein to cytosolusing a complex of nanoparticles and a cargo protein via endocytosis(Munish Chanana et al. Physicochemical properties of protein-coated goldnanoparticles in biological fluids and cells before and afterproteolytic digestion. Angew. Chem. Int. Ed. 2013).

FIG. 3 illustrates the process in which exosome is separated andreleased from multi-vesicular bodies (MVBs) (Graca Raposo and WillemStoorvogel. Extracellular vesicles: Exosomes, microvesicles, andfriends. Cell Biology 200(4), 373-383, 2013).

FIG. 4 illustrates the process of treating cancer by delivering siRNA invivo through the targeted exosome (Alvarez-Erviti, L. et al. Delivery ofsiRNA to the mouse brain by systemic injection of targeted exosomes.Nature Biotechnology 29, 341-345, 2011).

FIG. 5 illustrates the preparation process of optogenetically-designedprotein-carrying exosomes (EXPLORs) according to the present invention.

FIG. 6 illustrates the process of separating the fusion protein of acargo protein and a photo-specific binding protein in the inside ofexosome when the light irradiation on EXPLORs is stopped.

FIG. 7 illustrates the changes in the intracellular location of mCherryprotein according to the blue light irradiation in the transformedHEK293T cells introduced with CIBN-EGFP-CD9 gene and mCherry-CRY2 gene.

FIG. 8 illustrates the experimental procedure of obtaining EXPLORsaccording to the present invention.

FIG. 9 illustrates the results of measuring the changes of the contentof a cargo protein (mCherry protein) captured in exosome according tothe intensity of blue light.

FIG. 10 illustrates the results of investigation of the introduction ofa cargo protein in target cells after treating the target cells (HT1080)with exosome containing the cargo protein (mCherry), wherein the leftindicates the target cells not-treated with exosome and the rightindicates the target cells treated with exosome.

FIG. 11 is a set of a fluorescence image (a) illustrating the results ofinvestigation of the introduction of a cargo protein in target cellsafter treating the target cells (HT1080) with exosome containing thecargo protein (mCherry); and a graph (b) illustrating the results ofcomparison of the ratio of apoptotic cells induced by the treatment ofexosome.

FIG. 12 illustrates the changes in the intracellular location of mCherryprotein according to the blue light irradiation in the transformedHEK293T cells introduced with GIGANTEA-EGFP-CD9 gene andmCherry-FKF1LOV.

FIG. 13 illustrates the expression of the Luciferase-mCherry fusionprotein measured by fluorescence imaging (a) and the luciferase activityand the number of molecules in the production cells (b):

-   -   Control: HEK293T cells treated with nothing;    -   OVER: HEK293T cells introduced with Luciferase-mCherry-CRY2        alone;    -   XP: HEK293T cells introduced with XPACK-Luciferase-mCherry by        using XPACK (Systems Biosciences), the commercial vector        designed for exosome loading technique;    -   EXPLOR: HEK293T cells introduced with Luciferase-mCherry-CRY2        and CIBN-EGFP-CD9 according to the present invention.

FIG. 14 illustrates the luciferase activity (a) and the number ofmolecules (b) in the produced exosome:

-   -   NEG: exosome produced in the HEK293T cells treated with nothing;    -   OVER: exosome produced in the HEK293T cells introduced with        Luciferase-mCherry-CRY2;    -   XP: exosome produced in the HEK293T cells introduced with        XPACK-Luciferase-mCherry by using XPACK (Systems Biosciences),        the commercial vector designed for exosome loading technique;    -   EXPLOR: exosome produced in the HEK293T cells introduced with        Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the        present invention;    -   ON: exosome produced by culturing under the irradiation of 200        μW blue light for 72 hours,    -   OFF: exosome produced by culturing under the light-free        condition for 72 hours.

FIG. 15 illustrates the loading efficiency of a cargo protein in theexosome produced above.

FIG. 16 illustrates the transfer efficiency of a cargo protein into thetarget cells (HeLa) using exosome:

-   -   Control: exosome produced in the HEK293T cells treated with        nothing;    -   OVER: exosome produced in the HEK293T cells introduced with        Luciferase-mCherry-CRY2;    -   XP: exosome produced in the HEK293T cells introduced with        XPACK-Luciferase-mCherry by using XPACK (Systems Biosciences),        the commercial vector designed for exosome loading technique;    -   EXPLOR: exosome produced in the HEK293T cells introduced with        Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the        present invention;    -   ON: exosome produced by culturing under the irradiation of 200        μW blue light for 72 hours,    -   OFF: exosome produced by culturing under the light-free        condition for 72 hours.

FIG. 17 illustrates the location of the expression ofLuciferase-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicatingthey share the same position for the expression.

FIG. 18 illustrates the location of the expression of Cre-mCherry-CRY2and CIBN-EGFP-CD9 in HEK293T cells, indicating they share the sameposition for the expression.

FIG. 19A illustrates that treatment with Cre:EXPLOR induced theexpression of ZsGreen in HT1080 cells transiently transfected withpCAG-loxP-STOP-loxP-ZsGreen (Scale bars, 40 μm):

-   -   Negative: EXPLOR: no cre-loaded exosome as negative control;    -   Cre: EXPLOR: Cre-loaded exosome; and    -   pcMV-Cre: pCMV-Cre vector transfection as positive control.

FIG. 19B illustrates that treatment with Cre: EXPLOR induced theexpression of ZsGreen in HeLa cells transiently transfected withpCAG-loxP-STOP-loxP-ZsGreen (Scale bars, 40 μm):

-   -   Negative: EXPLOR: no Cre-loaded exosome as negative control;    -   Cre: EXPLOR: Cre-loaded exosome; and    -   pcMV-Cre: pCMV-Cre vector transfection as positive control.

FIG. 20 illustrates that treatment with Cre:EXPLOR induced theexpression of ZsGreen in primary rat embryonic neuron transientlytransfected with pCAG-loxP-STOP-loxP-ZsGreen (Scale bars, 100 μm):

-   -   Control: EXPLOR: no Cre-loaded exosome as negative control; and    -   Cre: EXPLOR::Cre-loaded exosome.

FIG. 21 illustrates that treatment with Cre:EXPLOR induced theexpression of ZsGreen in transgenic mice havingpCAG-loxP-STOP-loxP-eNpHR3.0-EYFP gene (Scale bars, 500 μm):

-   -   Control: EXPLOR: no Cre-loaded exosome as negative control;    -   Cre: EXPLOR: Cre-loaded exosome;    -   Hip: hippocampus; and    -   Th: thalamus.

FIG. 22 illustrates the results of immunohistochemistry for NEuN/GFAP intransgenic mice having pCAG-loxP-STOP-loxP-eNpHR3.0-EYFP gene

-   -   Pink: neuronal-specific nuclear protein; NEuN, positive neurons;        and    -   Red: glial fibrillary acidic protein; GFAP, positive astrocyte        cells.    -   Objective lens, 40×. Scale bar, 20 μm.

FIG. 23 illustrates the location of the expression of Cas9-mCherry-CRY2and CIBN-EGFP-CD9 in HEK293T cells, indicating they share the sameposition for the expression.

FIG. 24 illustrates the generation of DNA constructs used for theproduction of Cas9-loaded exosome.

FIG. 25 illustrates the results of measuring the content of a cargoprotein (CRISPR-Cas9 protein) captured in exosome.

FIG. 26 illustrates the location of the expression of GBA-mCherry-CRY2and CIBN-EGFP-CD9 in HEK293T cells, indicating they share the sameposition for the expression.

FIG. 27 illustrates the expression of endogenous GBA andGBA-mcherry-CRY2 fusion protein in HEK293T cell transiently transfectedwith GBA-mCh-CRY2 and CIBN-EGFP-CD9, rat primary astrocyte, humanprimary astrocyte and Gaucher fibroblast.

FIG. 28 illustrates the results of measuring the content of a cargoprotein (GBA protein) captured in exosome.

FIG. 29 illustrates the results of measuring the enzymatic activity ofβ-glucocerebrosidase, a cargo protein (GBA protein) captured in exosome.

-   -   Exo-Naive: HEK293T-derived exosome    -   Exo-GBA: exosome including β-glucocerebrosidase

FIG. 30 illustrates the results of treatment of GBA-exosomes to Gaucherdisease patient-derived fibroblasts, indicating treatment withGBA-exosomes significantly induced the enzymatic activity inβ-glucocerebrosidase-deficient cells.

FIG. 31 illustrates the generation of DNA constructs used for theproduction of PTEN-loaded exosome and cells stably expressingPTEN-loaded exosome.

FIG. 32 illustrates the location of the expression ofluciferase-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicatingthey share the same position for the expression.

FIG. 33 illustrates the results of measurement of quantitativeluciferase activity based on the number of luciferase molecules.

FIG. 34 illustrates the location of the expression ofPrxI/II-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating theyshare the same position for the expression.

-   -   Prx I: peroxiredoxin I    -   Prx II: peroxiredoxin II

FIG. 35 illustrates the protective effect of PrxI/II-loaded exosomes inH₂O₂-induced oxidative stress and cytotoxicity.

-   -   None: H₂O₂-treated group;    -   Cre: EXPLOR: Cre-loaded exosome    -   Prx I: EXPLOR: PrxI-loaded exosomes; and    -   Prx II: EXPLOR: PrxII-loaded exosomes.

FIG. 36 illustrates the location of the expression of MyoD-mCherry-CRY2and CIBN-EGFP-CD9 in HEK293T cells, indicating they share the sameposition for the expression.

FIG. 37 illustrates the results of treatment of MyoD-loaded exosomes toadipose-derived stem cells and, indicating treatment with MyoD-exosomes(clone #A6) induced the proliferation of cells after 6 days.

FIG. 38 illustrates the generation of cells stably expressing p53-loadedexosome.

FIG. 39 illustrates the results of measuring the content of a cargoprotein (p53 protein) captured in exosome.

-   -   Stable cell: cells stably expressing p53-loaded exosome.    -   mCherry: mCherry-loaded exosome    -   p53: p53-loaded exosome

FIG. 40 illustrates the results of measurement of transcriptionalactivity of p53 using luciferase reporter gene, indicating treatmentwith p53-loaded exosomes induced transcriptional activity of p53 indoxorubicin-treated HeLa cells.

FIG. 41 illustrates the generation of DNA constructs used for theproduction of HMGB1-loaded exosome and cells stably expressingHMGB1-loaded exosome.

FIG. 42 illustrates the location of the expression of srIκB-mCherry-CRY2and CIBN-EGFP-CD9 in HEK293T cells, indicating they share the sameposition for the expression.

FIG. 43 illustrates that treatment with srIkB-mCherry:EXPLORssignificantly reduced tumor necrosis factor-α-induced translocation andDNA binding of the p65 subunit of NF-κB in HeLa cells.

FIG. 44 illustrates the analysis of disease progression afteradministration of srIkB-loaded exosomes to rheumatoid arthritis animalmodel.

FIG. 45 illustrates the survival curve of groups treated withsrIkB-loaded exosomes in LPS-induced sepsis model.

-   -   No exosome: only LPS treated group    -   Naive exosome: group treated with HEK293T-derived exosome    -   srIkB exosome: group treated with srIkB-loaded exosomes

FIG. 46 illustrates the location of the expression of pYSTAT3intrabody-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicatingthey share the same position for the expression.

FIG. 47 illustrates the intracellular delivery of pYSTAT3 intrabody totarget cells using pYSTAT3 intrabody-loaded exosomes.

FIG. 48 illustrates location of the expression of Bax-mCherry-CRY2 andCIBN-EGFP-CD9 in HEK293T cells, indicating they share the same positionfor the expression.

FIG. 49 illustrates that treatment with Bax-loaded exosome induced arapid release of cytochrome c from the mitochondria in HeLa cells

FIG. 50 illustrates location of the expression of AIMP-mCherry-CRY2 andCIBN-EGFP-CD9 in HEK293T cells, indicating they share the same positionfor the expression.

FIG. 51 illustrates the results of measuring the content of a cargoprotein (AIMP protein) captured in exosome.

FIG. 52 illustrates location of the expression of mCherry-CRY2 andCIBN-EGFP-CD9 in HEK293T cells, indicating they share the same positionfor the expression.

FIG. 53 illustrates DNA deletion by Cre recombinase.

FIG. 54 illustrates difference between Cas9 and Cpf1 protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions containing exosome loadedwith a cargo protein.

In another embodiment, the present invention provides a method forpreparing the exosome loaded with a cargo protein using a photo-specificbinding protein.

In a further embodiment, the present invention provides a method ofdelivering the cargo protein to cytosol using the exosome.

In another embodiment, the present invention provides a method for themass-production of exosome containing a fusion protein composed of anexosome specific marker and a cargo protein.

The present invention provides a method for the mass-production ofexosome containing a cargo protein separated from the membrane ofexosome by using a photo-specific binding protein pair.

The present invention also provides a vector for preparing exosome whichis usable for the preparation of the exosome.

The present invention further provides a method to introduce a cargoprotein in cytosol by using the exosome above.

In one embodiment, the present invention provides pharmaceuticalcompostions containing exosomes loaded with a cargo proteins and amethod for preparing the same.

In a preferred embodiment, the cargo protein is super-repressor-IκBprotein inhibiting NF-κB, Bax(Bcl-2-associated X protein), PeroxiredoxinI, Peroxiredoxin II, Cre recombinase, Cas9 (CRISPR associated protein9), Cpf1(CRISPR from Prevotella and Francisella 1) orGBA(β-glucocerebrosidase).

The present invention provides exosome comprising a cargo protein whichcan be used for the treatment of various diseases in vivo by deliveringthe cargo protein. For example, exosome can be prepared to include aprotein or siRNA having an anticancer activity and then treated tocancer cells for cancer treatment (FIG. 4).

For the exosomes containing a cargo protein used for the treatment ofdisease, the exosomes needs to be prepared efficiently to have properload of the cargo protein. Korean Patent Publication No. 2004-0015508describes a method for preparing exosome comprising a specific antigen.Precisely, it describes a method of discharging a cargo protein by usingexosome, wherein a gene encoding a specific antigen is inserted in ahost cell line and a protein of the introduced gene is stably expressedin the cell line which is discharged extracellularly through exosome,and a method using the exosome as a vaccine.

However exosome is formed naturally within the cells. So, even though agene encoding a cargo protein is inserted in the cell producing exosomeendogenously, it is very difficult to prepare exosome comprising theexpressed protein in it thereby.

The present invention provides methods for preparing exosome comprisinga cargo protein more efficiently. As a result, the inventors succeededin preparing exosome comprising a cargo protein efficiently byexpressing a fusion protein composed of an exosome specific marker and acargo protein massively in the cell producing exosome endogenously at ahigh concentration (FIG. 5).

The cargo protein is attached on the membrane of exosome, according tothe method above. So, the fusion protein composed of a pair of anexosome specific marker and a cargo protein is expressed in the cellproducing exosome at a high concentration, followed by irradiation toinduce the linkage of the fusion protein. Then, the fusion protein isintroduced inside the exosome by the action of the exosome specificmarker. When the irradiation is terminated after the introduction, thefusion protein is separated into a cargo protein and a photo-specificbinding protein inside the exosome. As a result, the exosome containinga free cargo protein separated from the fusion protein can be preparedefficiently (FIG. 6).

The cargo proteins loaded in the exosome in the present inventionincludes, but not limited to, natural or non-natural proteins, truncatedform or mutated form. Examples of the cargo proteins are listed, but notlimited to, in the following table.

TABLE 1 Classification Sub-Class Example Enzymes Proteases(extracellular & MMPs and TIMP (tissue inhibitor intracellular) andtheir metalloproteases) inhibitors Caspases and their inhibitorsCathepsins and their inhibitors Nucleases Cre recombinase CRISPR/cas9Caspase-activated DNase hydrolytic enzymes Lysosomal enzymes includingBeta- glucocerebrosidase Kinases and phosphatase Mitogen activatedkinases: p38 MAP kinase Inhibitor kappa B kinase (IKK) PTEN phosphataseJanus kinase others Ubiquitin ligase luciferase peroxiredoxinsTranscription Transcription factors and NF-kB/super repressor IkBfactors their inhibitors MyoD Tbx18 (T-box transcription factor 18) p53HMGB1 (High mobility group box 1 protein) Antibodies Antibodies andassociated pYSTAT3 intrabody peptides others unclassified Pro-apoptoticproteins: Bax Anti-apoptotic proteins: BcL-xL Multifunctional signalmolecules: AIMP (Aminoacyl-tRNA synthetase- interacting multifunctionalproteins) Fluorescent proteins (mCherry, GFP) Nucleic acid-bindingproteins (ex. RNPs)

<Enzymes>

Enzymes are biological catalytic molecules accelerating chemicalreactions in living organisms. Enzymes bind to their substrates andfacilitate the reaction rate by lowering its activation energy. Enzymescan be classified as follows; proteases, nucleases, hydrolytic enzymes,kinases, phosphatase and other types of enzymes.

The target proteins loaded in the exosomes in the present inventioninclude enzymes and their regulators. Examples of the target proteinsare listed, but not limited to, in the following description.

Proteases and their Inhibitors

MMPs and TIMP

Matrix metalloproteinases (MMPs), also known as matrixins, arecalcium-dependent zinc-containing endopeptidases. MMPs are capable ofdegrading all kinds of extracellular matrix proteins and known to beinvolved in the cleavage of cell surface receptors, the release ofapoptotic ligands (such as the FAS ligand), and chemokine/cytokineinactivation. MMPs are also thought to play a major role in cellbehaviors such as cell proliferation, migration (adhesion/dispersion),differentiation, angiogenesis, apoptosis, and host defense.

The matrix metalloproteinases are inhibited by specific endogenoustissue inhibitors of metalloproteinases (TIMPs), which comprise a familyof four protease inhibitors: TIMP1, TIMP2, TIMP3 and TIMP4.

The balance of MMPs and TIMPs plays an important role in tissueremodeling associated with various physiological or pathologicalprocesses such as morphogenesis, angiogenesis, tissue repair, cirrhosis,arthritis, and metastasis. MMP-2 and MMP-9 are thought to be importantin metastasis. MMP-1 is thought to be important in rheumatoid arthritisand osteoarthritis. Dysregulation of the balance between MMPs and TIMPsis also a characteristic of acute and chronic cardiovascular diseases.

The exosomes comprising MMPs and TIMPs are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with MMPs or TIMPs can be used totreat MMP-associated diseases including rheumatoid arthritis.

Caspases and their Inhibitors

Caspases (cysteine-aspartic proteases, cysteine aspartases orcysteine-dependent aspartate-directed proteases) are a family ofprotease enzymes playing essential roles in programmed cell deathincluding apoptosis, pyroptosis and necroptosis. These forms of celldeath are important for protecting an organism from stress signals andpathogenic attack. Caspases also have a role in inflammation, whereby itdirectly processes pro-inflammatory cytokines such as pro-IL1β. Theseare signaling molecules that allow recruitment of immune cells to aninfected cell or tissue. There are other identified roles of caspasessuch as cell proliferation, tumor suppression, cell differentiation,neural development and axon guidance and ageing.

Caspase deficiency has been identified as a cause of tumor development.Tumor growth can occur by a combination of factors, including a mutationin a cell cycle gene which removes the restraints on cell growth,combined with mutations in apoptotic proteins such as Caspases thatwould respond by inducing cell death in abnormally growing cells.

Conversely, over-activation of some caspases such as caspase-3 can leadto excessive programmed cell death. This is seen in severalneurodegenerative diseases where neural cells are lost, such asAlzheimer's disease. Caspases involved with processing inflammatorysignals are also implicated in disease. Insufficient activation of thesecaspases can increase an organism's susceptibility to infection, as anappropriate immune response may not be activated. The integral rolecaspases play in cell death and disease has led to research on usingcaspases as a drug target. For example, inflammatory caspase-1 has beenimplicated in causing autoimmune diseases.

The exosomes comprising caspases and their inhibitors are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with caspases or their inhibitors canbe used to treat caspase-associated diseases including neurodegenerativediseases or autoimmune diseases.

Cathepsins and their Inhibitors

Cathepsins are proteases found in all animals as well as otherorganisms. There are approximately a dozen members of this family, whichare distinguished by their structure, catalytic mechanism, and whichproteins they cleave. Most of the members become activated at the low pHfound in lysosomes. Thus, the activity of this family lies almostentirely within those organelles.

Cathepsins have been implicated in cancer, stroke, Alzheimer's disease,arthritis, Ebola, COPD, chronic periodontitis, pancreatitis and severalocular disorders including keratoconus. Especially for cancer, cathepsinD is a mitogen and it attenuates the anti-tumor immune response ofdecaying chemokines to inhibit the function of dendritic cells.Cathepsin B and L are involved in matrix degradation and cell invasion.

The exosomes comprising cathepsins and their inhibitors are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with cathepsins or their inhibitorscan be used to treat varying cathepsin-associated diseases includingcancer and Alzheimer's disease.

Nucleases

Cre Recombinase

Cre recombinase is the protein isolated from P1 bacteriophage, andinduces recombination by detecting two different loxP region. The loxPis DNA fragment with 34 bp and is composed of two 13 bp palindromicsequence on both extremes and 8 bp asymmetrical core spacer on middle.The Cre recombinase binds to palindromic sequence, change the spacerregion of DNA after cutting, and then recombine DNA (FIG. 53). Excisionor inversion of DNA sequence between two different lowP regions based onthe directionality of spacer. The excision or inversion is occurred ifdirection of lowP region is same or reverse, respectively.

One of the representative examples of Cre recombinase utilization is theconditional knockout mouse which can inhibit mutated period andexpressed tissues of specific gene. This technology is that eliminatingspecific target gene in some isolated cells by producing loxP insertedmouse between front and end of specific target gene, mating withCre-expressing transgenic mouse, or directly treating Cre recombinase tospecific cell. The conditional knockout mouse is efficient to confirmthe function of specific gene by expressing such gene, which is lethalin early phase of embryo development, in late phase of embryodevelopment or adult.

The present invention provides exosomes loaded with Cre recombinaseprotein and confirmed that Cre recombinase protein was delivered to thecytosol of the target cells. The results indicate that the exosome ofthe present invention loaded with Cre recombinase protein can be usedfor conditional gene manipulation.

CRISPR/Cas9

CRISPR-Cas9 is an RNA-based artificial restriction enzyme that makes DNAcorrection be possible by restricting specific region of genes.Recently, it is remarkably spotlighted as the key element of geneticengineering.

CRISPR, which is kind of palindromic sequence, is the abbreviated formof Clustered regularly-interspaced short palindromic repeats and firstobserved acquired immunity system of bacterium. Firstly, Cas9 proteinrecognizes and restricts invaded virus. Then the restricted virussequence is inserted into CRISPR sequence, and combined virus and CRISPRsequence is transcribed as RNA. This RNA is used in formation of Cas9complex. After this process, transcribed ‘CRISPR+virus sequence’ iscombined with Cas9 and eliminates same invaded virus faster than Cas9alone. This mechanism can be applied in genetic engineering by combiningtarget sequence with Cas9 complex to restrict target sequence.

Cpf1 is protein with similar function with Cas9 protein fromaforementioned engineered endonuclease CRISPR-Cas9 system. As shown inFIG. 54, Cpf1 recognizes protospace adjacent motif (PAM) sequence unlikeCas9. It can be used on the region unrecognized by Cas9, and especiallyit is more practical because short crispr RNA (crRNA) alone can beworked. In case of Cas9, tracrRNA is additionally needed.

The present invention provides exosomes loaded with Cas9 or Cpf1 proteinand confirmed that Cas9 or Cpf1 protein was delivered to the cytosol ofthe target cells. The results indicate that the exosome of the presentinvention loaded with Cas9 or Cpf1 protein can be used for removing,adding or altering sections of the DNA sequence.

Caspase-Activated DNase

Caspase-Activated DNase (CAD) or DNA fragmentation factor subunit beta(DFFB) is a protein that is encoded by the DFFB gene in humans. Itbreaks up the DNA during apoptosis and promotes cell differentiation. Itis usually an inactive monomer inhibited by ICAD. This is cleaved beforedimerization.

Apoptosis is a cell death process that removes toxic and/or uselesscells during mammalian development. The apoptotic process is accompaniedby shrinkage and fragmentation of the cells and nuclei and degradationof the chromosomal DNA into nucleosomal units. DNA fragmentation factor(DFF) is a heterodimeric protein of 40-kD (DFFB) and 45-kD (DFFA)subunits. DFFA is the substrate for caspase-3 and triggers DNAfragmentation during apoptosis. DFF becomes activated when DFFA iscleaved by caspase-3. The cleaved fragments of DFFA dissociate fromDFFB, the active component of DFF. DFFB has been found to trigger bothDNA fragmentation and chromatin condensation during apoptosis.

The exosomes comprising Caspase-activated DNase are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with Caspase-activated DNase can beused to regulate apoptosis in diverse systems.

Hydrolytic Enzymes

Lysosomal Enzyme Including Beta-Glucocerebrosidase

Lysosomal storage disorder is the disease because of storage ofmaterials degraded by lysosome according to the innate deficiency oflysosome. One of the common lysosomal storage disorder is Gaucherdisease which is induced by genetic deficiency of β-glucocerebrosidase(GB A), lysosomal enzyme.

Lack of GBA induces malfunction on liver, spleen, and bone marrow, andso on by storing glucocerebrosidase/glucosylsphingosine on lysosome ofmacrophage. Also it induces hematologic abnormality such as anemia,thrombocytopenia, and leukopenia, gepatolientalny, osteoclasia, andcentral nerve injury, etc.

Present treatment of Gaucher disease is the enzyme replacement therapyinjecting GBA analogue, cerezyme, by intravenous injection. However,these kinds of protein drugs have various disadvantage such as shorthalf-life in blood, low efficiency because of antibody production,difficulty of delivery to lysosome, and the impossibility on applyingneurogenic Gaucher disease, etc.

The present invention provides exosomes loaded with GBA(β-glucocerebrosidase) protein and confirmed that GBA(β-glucocerebrosidase) protein was delivered to the cytosol of thetarget cells. The results indicate that the exosome of the presentinvention loaded with GBA (β-glucocerebrosidase) protein can be used fortreatment of Gaucher disease.

Kinases and Phosphatase

Mitogen Activated Kinases: p38 MAP Kinase

P38 mitogen-activated protein kinases are a class of mitogen-activatedprotein kinases (MAPKs) that are responsive to stress stimuli, such ascytokines, ultraviolet irradiation, heat shock, and osmotic shock. P38MAP kinase are involved in cell differentiation, apoptosis andautophagy.

P38 MAP Kinase (MAPK) participates in a signaling cascade controllingcellular responses to cytokines and stress. P38 inhibitors are beingsought for possible therapeutic effect on autoimmune diseases andinflammatory processes.

The exosomes comprising p38 MAPK and its inhibitor are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with p38 MAPK or its inhibitors canbe used to treat p38 MAPK-associated diseases including autoimmunediseases.

Inhibitor Kappa B Kinase (IKK)

The IκB kinase (IKK) is an enzyme complex that is involved inpropagating the cellular response to inflammation. The IκB kinase enzymecomplex is part of the upstream NF-κB signal transduction cascade. TheIκB a (inhibitor of kappa B) protein inactivates the NF-κB transcriptionfactor by masking the nuclear localization signals (NLS) of NF-κBproteins and keeping them sequestered in an inactive state in thecytoplasm. IKK phosphorylates the inhibitory IκB a protein. Thisphosphorylation results in the dissociation of IκBa from NF-κB. NF-κB,which is now free, migrates into the nucleus and activates theexpression of at least 150 genes; some of which are anti-apoptotic.

IκB kinase activity is essential for activation of members of thenuclear factor-kB (NF-κB) family of transcription factors, which play afundamental role in lymphocyte immune-regulation. Activation of thecanonical NF-κB pathway begins in response to stimulation by variouspro-inflammatory stimuli, including lipopolysaccharide (LPS) expressedon the surface of pathogens, or the release of pro-inflammatorycytokines such as tumor necrosis factor (TNF) or interleukin-1 (IL-1).Following immune cell stimulation, a signal transduction cascade leadsto the activation of the IKK complex, an event characterized by thebinding of NEMO to the homologous kinase subunits IKK-α and IKK-β.

Though functionally adaptive in response to inflammatory stimuli,deregulation of NF-κB signaling has been exploited in various diseasestates. Increased NF-κB activity as a result of constitutiveIKK-mediated phosphorylation of IκBα has been observed in thedevelopment of atherosclerosis, asthma, rheumatoid arthritis,inflammatory bowel diseases, and multiple sclerosis. Specifically,constitutive NF-κB activity promotes continuous inflammatory signalingat the molecular level that translates to chronic inflammationphenotypically. Furthermore, the ability of NF-κB to simultaneouslysuppress apoptosis and promote continuous lymphocyte growth andproliferation explains its intimate connection with many types ofcancer.

The exosomes comprising IKK are prepared by expressing a fusion proteincomposed of an exosome specific marker and a target protein massively inthe cell producing the exosomes at a high concentration. The exosomesloaded with IKK can be used to treat NF-κB-associated diseases includingcancers.

PTEN Phosphatase

Phosphatase and tensin homolog (PTEN) is identified as a tumorsuppressor protein. Mutations of this gene are a step in the developmentof many cancers. The protein contains a tensin-like domain as well as acatalytic domain similar to that of the dual specificity proteintyrosine phosphatases. Unlike most of the protein tyrosine phosphatases,this protein preferentially dephosphorylates phosphoinositidesubstrates. It negatively regulates intracellular levels ofphosphatidylinositol-3, 4, 5-trisphosphate in cells and functions as atumor suppressor by negatively regulating Akt/PKB signaling pathway.

PTEN loss or mutation is closely related with cancer, non-cancerousneoplasia and autism. Especially during tumor development, mutations anddeletions of PTEN occur that inactivate its enzymatic activity leadingto increased cell proliferation and reduced cell death. Frequent geneticinactivation of PTEN occurs in glioblastoma, endometrial cancer, andprostate cancer; and reduced expression is found in many other tumortypes such as lung and breast cancer. Furthermore, PTEN mutation alsocauses a variety of inherited predispositions to cancer.

Mutations in the PTEN gene cause several other disorders that, likeCowden syndrome, are characterized by the development of non-canceroustumors called hamartomas. These disorders includeBannayan-Riley-Ruvalcaba syndrome and Proteus-like syndrome. Together,the disorders caused by PTEN mutations are called PTEN hamartoma tumorsyndromes, or PHTS. Mutations responsible for these syndromes cause theresulting protein to be non-functional or absent. The defective proteinallows the cell to divide in an uncontrolled way and prevents damagedcells from dying, which can lead to the growth of tumors.

The exosomes comprising PTEN are prepared by expressing a fusion proteincomposed of an exosome specific marker and a target protein massively inthe cell producing the exosomes at a high concentration. The exosomesloaded with PTEN can be used to treat varying types of cancers.

Janus Kinase

Janus kinase (JAK) is a family of intracellular, nonreceptor tyrosinekinases that transduce cytokine-mediated signals via the JAK-STATpathway. Since members of the type I and type II cytokine receptorfamilies possess no catalytic kinase activity, they rely on the JAKfamily of tyrosine kinases to phosphorylate and activate downstreamproteins involved in their signal transduction pathways. After thereceptor associates with its respective cytokine/ligand, it goes througha conformational change, bringing the two JAKs close enough tophosphorylate each other. The JAK auto-phosphorylation induces aconformational change within itself, enabling it to transduce theintracellular signal by further phosphorylating and activatingtranscription factors called STATs (Signal Transducer and Activator ofTranscription). The activated STATs dissociate from the receptor andform dimers before translocating to the cell nucleus, where theyregulate transcription of selected genes.

Some examples of the molecules that use the JAK/STAT signaling pathwayare colony-stimulating factor, prolactin, growth hormone, and manycytokines. JAK inhibitors are under development for the treatment ofpsoriasis, rheumatoid arthritis, polycythemia vera, alopecia, essentialthrombocythemia, ulcerative colitis, myeloid metaplasia withmyelofibrosis and vitiligo.

The exosomes comprising JAK and its inhibitors are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with JAK or its inhibitors can beused to treat JAK-associated diseases including cancers.

Others

Ubiquitin Ligase

A ubiquitin ligase (also called an E3 ubiquitin ligase) is a proteinthat recruits an E2 ubiquitin-conjugating enzyme that has been loadedwith ubiquitin, recognizes a protein substrate, and assists or directlycatalyzes the transfer of ubiquitin from the E2 to the proteinsubstrate. The ubiquitin is attached to a lysine on the target proteinby an isopeptide bond. E3 ligases interact with both the target proteinand the E2 enzyme, and so impart substrate specificity to the E2.

Ubiquitination by E3 ligases regulates diverse areas such as celltrafficking, DNA repair, and signaling and is of profound importance incell biology. E3 ligases are also key players in cell cycle control,mediating the degradation of cyclins, as well as cyclin dependent kinaseinhibitor proteins.

E3 ubiquitin ligases regulate homeostasis, cell cycle, and DNA repairpathways, and as a result, a number of these proteins are involved in avariety of cancers, including famously MDM2, BRCA1, and VonHippel-Lindau tumor suppressor. For example, a mutation of MDM2 has beenfound in stomach cancer, renal cell carcinoma, and liver cancer (amongstothers) to deregulate MDM2 concentrations by increasing its promoter'saffinity for the Sp1 transcription factor, causing increasedtranscription of MDM2 mRNA.

The exosomes comprising Ubiquitin ligase are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with Ubiquitin ligase can be used totreat ubiquitination-associated diseases including cancers.

Luciferase

Luciferase is a generic term for the class of oxidative enzymes thatproduce bioluminescence, and is usually distinguished from aphotoprotein. Luciferases are widely used in biotechnology, formicroscopy and as reporter genes, for many of the same applications asfluorescent proteins. However, unlike fluorescent proteins, luciferasesdo not require an external light source, but do require addition ofluciferin, the consumable substrate.

All luciferases are classified as oxidoreductases (EC 1.13.12.-),meaning they act on single donors with incorporation of molecularoxygen. Because luciferases are from many diverse protein families thatare unrelated, there is no unifying mechanism, as any mechanism dependson the luciferase and luciferin combination. However, all characterizedluciferase-luciferin reactions to date have been shown to requiremolecular oxygen at some stage.

In biological research, luciferase is commonly used as a reporter toassess the transcriptional activity in cells that are transfected with agenetic construct containing the luciferase gene under the control of apromoter of interest. Additionally, pro-luminescent molecules that areconverted to luciferin upon activity of a particular enzyme can be usedto detect enzyme activity in coupled or two-step luciferase assays. Suchsubstrates have been used to detect caspase activity and cytochrome P450activity, among others. Luciferase can also be used to detect the levelof cellular ATP in cell viability assays or for kinase activity assays.Luciferase can act as an ATP sensor protein through biotinylation.Biotinylation will immobilize luciferase on the cell-surface by bindingto a streptavidin-biotin complex. This allows luciferase to detect theefflux of ATP from the cell and will effectively display the real-timerelease of ATP through bioluminescence. Luciferase can additionally bemade more sensitive for ATP detection by increasing the luminescenceintensity by changing certain amino acid residues in the sequence of theprotein.

Whole animal imaging (referred to as in vivo or, occasionally, ex vivoimaging) can be performed using luciferase-expressing cell lineinjection. Different types of cells (e.g. bone marrow stem cells,T-cells) can be engineered to express a luciferase allowing theirnon-invasive visualization inside a live animal using a sensitivecharge-couple device camera (CCD camera). This technique has been usedto follow tumorigenesis and response of tumors to treatment in animalmodels.

The present invention prepared exosomes loaded with luciferase proteinand confirmed that luciferase protein was delivered to the cytosol ofthe target cells. The results indicate that the exosome of the presentinvention loaded with luciferase protein can be used for cell viabilityassay, kinase activity assay and whole animal imaging.

Peroxiredoxins

Peroxiredoxin (Prx) is representative antioxidant enzyme in cytoplasmand obtain 0.1˜0.8% of water-soluble protein in mammalian cells. Prx hasthe role to reduce hydroperoxide to H₂O and ROH— by receiving 2e− incells. Prx also involves in cell proliferation, differentiation, death,and cell signal transduction by participating the formation andelimination of H₂O₂ (nmol concentration). Prx is classified morespecifically into 1-Cys Prx, or 2-Cys Prx based on the number ofcysteine amino acid. Furthermore, 2-Cys prx is subdivided into ‘typical’or ‘atypical’ based on structural, and mechanistic difference. All threePrx have difference in oxidation-reduction from second process offormation of Cys-SOH. Prx I-Prx IV are typical 2-Cys Prx, and Prx V isatypical 2-Cys Prc, and Prx VI is 1-Cys Prx. Some cases of 2-Cys Prxform oligomer.

Prx I, and II involve in activation of receptor-signaling pathway byregulating the concentration of H₂O₂ in cell generated by growth factorand TNF-α. Specifically, Prx II has the role in protecting cells fromstimulus of cell-death inducing factor such as serum starvation,ceramide, and etoposide.

In normal cells, Prx I have the role to maintain activity of PTENphosphatase by inhibiting its oxidation. However, in case of increasedoxidative stress, the activity of PTEN is inhibited by H₂O₂ throughseparation of Prx from PTEN by irreversible oxidation. Consequently, itinduces tumor through continuous activation of cell proliferating signalsuch as Akt.

It has a significant relation with disease that quantitative change ofPrx in cell. During the process of cancer development, arteriosclerosis,respiratory inflammation, osteoporosis, obesity, and degenerativedementia, quantitative change of reactive oxygen species has a closeconnection.

The present invention provides exosomes loaded with Peroxiredoxin I orPeroxiredoxin II protein and confirmed that Peroxiredoxin I orPeroxiredoxin II protein was delivered to the cytosol of the targetcells. The results indicates that the exosome of the present inventionloaded with Peroxiredoxin I or Peroxiredoxin II protein can be used fortreatment of reactive oxygen-related diseases.

<Transcription Factors>

Transcription factors are proteins regulating mRNA transcription fromDNA in eukaryotes. Transcription factors are associated with the basaltranscription regulation, organism development, response tointercellular signals or environment, cell cycle control andpathogenesis.

The target proteins loaded in the exosomes in the present inventioninclude transcription factors and their regulators (enhancers orinhibitors). Examples of the target proteins are listed, but not limitedto, in the following description.

Transcription Factors and their Regulators

NF-kB Regulator, Super-Repressor IkB

NF-κB is the major transcription factor inducing the inflammatoryresponse, and regulates the expression of inflammatory-related genes invarious types of cells especially immune cells. Therefore, it can beeffective therapeutic strategy for incurable chronic inflammatorydisease such as rheumatoid arthritis, sepsis, and psoriasis thatselectively inhibits the overactive NF-κB signaling pathway in immunecells. In addition, activation of NF-κB has the role that inhibitsapoptosis by increasing the expression of anti-apoptotic factors. Fromthis role, continuous activation of NF-κB signaling pathway in cancer isthe cause for anticancer drug resistance and then decreases thetherapeutic effects of anticancer drugs.

Most NF-κB is on inactive phase by binding with IκB, which is theinhibitory protein of NF-κB, in normal cells. IκB Kinase (IKK) complexactivated by various stimuli such as TNF-α and LPS phosphorylates IκB.The phosphorylated IκB is then ubiquitinated and finally degraded byproteasome. Through degradation of IκB, NF-κB (p50/p65) bound on IκBpasses through nuclear membrane. After passing, it activates mRNAtranscription by binding on the promotor region of target genes innucleus. This is the important element of immune response that inducestranscription of cytokine and inflammatory mediator such as iNOS, COX-2,NO, PGE2, TNF-α, and IL-1 (Lappas et al., Biol. Reprod. 67:668673,2002).

Super-repressor IκB which is S32A and S36A mutant form of IκB cancontinuously inhibit NF-κB because it is not phosphorylated by IκBKinase and degraded by proteasome. Therefore, it has the great potentialas treatment for various inflammatory diseases.

The present invention provides exosomes loaded with Super-repressor IκBprotein and confirmed that Super-repressor IκB protein was delivered tothe cytosol of the target cells. The results indicate that the exosomeof the present invention loaded with Super-repressor IκB protein can beused for treatment of inflammatory diseases.

MyoD

MyoD is a protein that plays critical role in regulating muscledifferentiation. MyoD belongs to a family of proteins known as myogenicregulatory factors (MRFs). MyoD is known to have binding interactionswith hundreds of muscular gene promoters and to permit myoblastproliferation. Also, one of the main functions of MyoD is to removecells from the cell cycle by enhancing the transcription of p21 andmyogenin.

The exosomes comprising MyoD protein are prepared by expressing a fusionprotein composed of an exosome specific marker and a target proteinmassively in the cell producing the exosomes at a high concentration.The exosomes loaded with MyoD can be used to treat myoblast-associateddiseases.

Tbx18 (T-Box Transcription Factor 18)

Tbx18 codes for a member of an evolutionarily conserved family oftranscription factors that plays a crucial role in embryonicdevelopment. Tbx18 is characterized by the presence of the DNA-bindingT-box domain and it belongs to the vertebrate specific Tbx1 sub-family.Tbx18 acts as a transcriptional repressor by antagonizingtranscriptional activators in the T-box family. Tbx18 is required invarious developmental process in tissues and organs, including the heartand coronary vessels, the ureter and the vertebral column. It is alsorequired for sinoatrial node (SAN) head area.

Tbx18 transduction is a method of turning on genes in heart muscle cellsas a treatment for certain cardiac arrhythmias. In a healthy heart,sinoatrial nodal cells act as the heart's pacemaker and cause the heartto beat in a regular rhythm. The problem in sick sinus syndrome is thatSA node is not functioning properly and is causing an irregularheartbeat. Expression of Tbx18 using adenovirus into atrial myocytesconverts atrial muscle cells into SA node cells that initiate theheartbeat. Tbx18 can be a one of many forms of gene therapy that cancure cardiac arrhythmias.

The exosomes comprising Tbx18 protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with Tbx18 protein can be used forthe treatment of sick sinus syndrome.

p53

Tumor protein p53 is known as the guardian of the genome because itconserves the stability of genome by preventing genome mutation. p53 canactivate DNA repair proteins when DNA has sustained damage. In addition,p53 can arrest growth by holding the cell cycle at the G1/S regulationpoint on DNA damage recognition. Upon DNA damage and it is irreparable,p53 can induce apoptosis. Lastly, p53 is essential for the senescenceresponse to short telomeres. p53 becomes activated in response to myriadstressors, including DNA damage, oxidative stress, osmotic shock,ribonucleotide depletion and deregulated oncogene expression.

If the p53 is damaged, tumor suppression is severely compromised. Peoplewho inherit only one functional copy of p53 gene will most likelydevelop tumors in early adulthood. Increasing the amount of p53 may seema solution for treatment of tumors or prevention of their spreading.

The exosomes comprising p53 protein are prepared by expressing a fusionprotein composed of an exosome specific marker and a target proteinmassively in the cell producing the exosomes at a high concentration.The exosomes loaded with p53 protein can be used for the treatment ofvarying types of cancers.

HMGB1 (High Mobility Group Box 1 Protein)

HMGB1 is among the most important chromatin proteins like histones. Inthe nucleus, HMGB1 interacts with nucleosomes, transcription factors andhistones. This nuclear protein organizes the DNA and regulatestranscriptions. After binding, HMGB1 bends DNA, which facilitates thebinding of other proteins. It also interacts with nucleosomes to loosenpacked DNA and remodel the chromatin.

HMGB1 is secreted by immune cells through leaderless secretory pathway.Activated macrophages and monocytes secrete HMGB1 as a cytokine mediatorof inflammation. Antibodies that neutralize HMGB1 confer protectionagainst damage and tissue injury during arthritis, colitis, ischemia,sepsis, etc.

The exosomes comprising HMGB1 protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with HMGB1 protein can be used forthe treatment of inflammatory diseases.

NeuroD1

Neurogenic differentiation1, also called β2, is a transcription factorof the NeuroD-type. It mediates transcriptional activation by binding toE box-containing promoter consensus core sequences 5′-CANNTG-3′. It iscontributed to the regulation of several cell differentiation pathways.It promotes the formation of early retinal ganglion cells, inner earsensory neurons and granule cells forming either the cerebellum or thedentate gyrus cell layer of the hippocampus, endocrine islet cells ofthe pancreas and enteroendocrine cells of the small intestine.

The exosomes comprising NeuroD1 protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with NeuroD1 protein can be used forthe regulation of neuron development.

Tumor-associated macrophages (TAMs) are a type of cell belonging to themacrophage lineage. They are found in close proximity or within tumormasses. TAMs are derived from circulating monocytes or resident tissuemacrophages, which form the major leukocytic infiltrate found within thestroma of many tumor types. TAMs have been linked to poor prognosis inbreast cancer, ovarian cancer, types of glioma and lymphoma; betterprognosis in colon and stomach cancers and both poor and betterprognoses in lung and prostate cancers.

TAMs are classified into two major phenotypes, M1 and M2. M1 TAMssuppress cancer progression, while M2 TAMs promote it. Severaltranscription factors are associated with the transition of M2macrophage to M1 macrophage. The target proteins loaded in the exosomesin the present invention include transcription factors associated withthe M2 to M1 conversion of macrophage. Examples of the target proteinsare listed, but not limited to, in the following description.

IRF5

IRF5 is a member of the interferon regulatory factor, a group oftranscription factor. It has role in virus-mediated activation ofinterferon and modulation of cell growth, differentiation, apoptosis andimmune system activity. IRF5 work by directly interacting with DNA orwith other proteins.

IRF5 acts as a molecular switch that controls whether macrophages willpromote or inhibit inflammation. Blocking the production of IRF inmacrophage can help treat a wide range of autoimmune disease andupregulating IRF5 levels can help treat people whose immune system areweak or damaged.

The exosomes comprising IRF5 protein are prepared by expressing a fusionprotein composed of an exosome specific marker and a target proteinmassively in the cell producing the exosomes at a high concentration.The exosomes loaded with IRF5 protein can be used for macrophagetransition from M2 to M1 for the treatment of varying types of cancers.

IRF3

IRF3 is a member of the interferon regulatory factors, a group oftranscription factor. IRF3 includes functional domains, nuclear exportsignal, a DNA-binding domain, a C-terminal IRF association domain andseveral regulatory sites. It is found in an inactive form in thecytoplasm of uninfected cells. Upon viral infection, double stranded RNAor toll-like receptor signaling, it is phosphorylated by IKBKE and TBK1kinases. This leads to dimerization and nuclear localization. IRF3 canactivate distinct gene expression programs in macrophages.

The exosomes comprising IRF3 protein are prepared by expressing a fusionprotein composed of an exosome specific marker and a target proteinmassively in the cell producing the exosomes at a high concentration.The exosomes loaded with IRF3 protein can be used for macrophagetransition from M2 to M1 for the treatment of varying types of cancers.

STAT1

Signal transducer and activator of transcription 1 is a transcriptionfactor, member of the STAT protein family. STAT1 can be activated byseveral ligands such as interferon alpha, interferon gamma, epidermalgrowth factor, platelet derived growth factor or interleukin 6.

Following type I IFN binding to cell surface receptors, JAK getsactivated and phosphorylates STAT1 and STAT2. STATs dimerize andassociate with ISGF3G/IRF-9 to form a complex termed ISGF3 transcriptionfactor. ISGF3 binds to the IFN stimulated response element to activatethe transcription of IFN-stimulated genes.

In response to type II IFN, STAT1 is tyrosine and serine phosphorylates.It forms a homodimer and binds to IFN gamma activated sequence to drivethe expression of target genes, inducing a cellular antiviral state.

The exosomes comprising STAT1 protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with STAT1 protein can be used forthe treatment of varying types of cancers.

SOCS3

Suppression of cytokine signaling is a member of STAT-induced STATinhibitor. STAT-induced STAT inhibitors are cytokine-inducible negativeregulators of cytokine signaling. SOCS3 is induced by various cytokineslike IL6, IL10, and IFN-gamma.

Overexpression of SOCS3 inhibits insulin signaling in adipose tissue andliver but not in muscle. But deletion of SOCS3 in the skeletal muscle ofmice protects against the obesity. SOCS3 also contributes to both leptinresistance and insulin resistance as a result of increased ceramidesynthesis. Study shows that removal of the SOCS gene prevents againstinsulin resistance in obesity. SOCS3 protein can bind to JAK2 andinhibits the activity of JAK2.

The exosomes comprising SOCS3 protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with SOCS3 protein can be used forthe treatment of varying types of cancers.

<Antibodies>

Antibodies are the proteins that recognize and bind to their specificantigen via the Fab's variable region on the tip of the “Y”-shapedantibody. Antibodies can suppress the activity of the target antigenproteins by binding to them.

The target proteins loaded in the exosomes in the present inventioninclude antibodies and antibody-associated peptides. Examples of thetarget proteins are listed, but not limited to, in the followingdescription.

Antibodies and Associated Peptides

pYSTAT3 Intrabody

STATs (Signal Transducer and Transcriptions) are transcription factorsthat have been identified: STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A andSTAT5B), and STAT6. STAT3 proteins have C-terminal transactivationdomain (Tyrosine 705 and Serine 727 residues for the majorphosphorylation sites of STAT3). Tyrosine phosphorylation and subsequentdimerization of STAT3 promote the transportation to the nucleus andtranscriptional activation.

The JAK/STAT3 signaling pathway is identified in growth factor-inducedactivation of interferon signaling and involved in proliferation,differentiation, apoptosis, angiogenesis, oncogenesis and immunity.Therefore, STAT3 proteins can be a good target as a single agent orcombination therapeutics for development of anticancer drugs.

The exosomes of the present invention loaded with pYSTAT3 intrabody wasprepared and confirmed that pYSTAT3 intrabody was delivered to thecytosol of the target cells. The results indicates that the exosome ofthe present invention loaded with pYSTAT3 intrabody can be used fortreatment of cancer.

<Others>

Apoptosis-Associated Proteins

Apoptosis (programmed cell death) is the process for eliminating damagedcells by various factors and abnormal apoptosis induces tumorigenicity.Based on this reason, researches about inducing apoptosis of tumor haveactively progressed as tumor-eliminating strategy. Condensation ofchromatin by cell atrophy, apoptotic body formation, and DNAfragmentation are the features of apoptosis. The apoptosis is induced bytwo different routes; one is the intrinsic pathway through mitochondria,and the other is the extrinsic pathway through death receptors. Theapoptosis is regulated variously, for example activation ofpro-apoptotic Bcl-2 family, segmentation of pro-caspase, andfragmentation of poly ADP-ribose polymerase (PARP), and so on.Especially caspases belonged to cysteine proteases are being pro-enzymein normally proliferated cells and activated by apoptotic inducingsignals, then has the significant role in apoptosis through involvingcargo proteins such as PARP.

Most apoptotic stimuli induce the apoptosis of mammalian cells throughthe pathway controlled by members of Bcl-2 gene family which are codinghomologous protein group including agonist and antagonist of apoptosissuch as Bcl-2, and Bcl-xL. These members share the sequence homologousdomain even though they are regulated discriminately. During apoptosis,the anti-apoptotic or pro-apoptotic effect of Bcl-2 and Bax (21%identity with Bcl-2 at the protein level) is regulated by homo- andheterodimers, which are differently formed by the ratio of Bcl-2 to Bax.

Pro-Apoptotic Proteins: Bax

Bax (Bcl-2-associated X protein) is the one of Bcl-2 protein family,so-called Bcl-2 like protein 4. Aforementioned Bax, which binds to theexternal membrane of mitochondria and its 4 residues of C-terminalprotrude on intermembrane space of mitochondria, has the role toactivate apoptosis. Specific information about aforementioned proteinand base sequence of its gene is noticed on NCBI (GenBank: NM_001291428,NP_001278357, etc.).

Bax is the one of Bcl-2 gene family synthesizing pro-apoptotic protein.Bax is inhibited its transcription by mutant p53. It has well known thatinsertion or deletion of Bax base sequence is the cause of markedlydecreased expression of Bax in cell lines of blood, colon, and rectalcancer.

It is known that Bax involves in apoptosis of neuron in development,homeostatic equilibrium of lymphatic and genital system, cell death byDNA damage, damage of ischemia reperfusion and so on.

The present invention provides exosomes loaded with Bax protein andconfirmed that Bax protein was delivered to the cytosol of the targetcells. The results indicates that the exosome of the present inventionloaded with Bax protein can be used for treatment of cancer.

Anti-Apoptotic Proteins: Bcl-xL

B-cell lymphoma-extra-large (Bcl-xL), encoded by the BCL2-like 1 gene,is a transmembrane molecule in the mitochondria. It is a member of theBcl-2 family of proteins, and acts as an anti-apoptotic protein bypreventing the release of mitochondrial contents such as cytochrome c,which leads to caspase activation and ultimately, programmed cell death.

It is a well-established concept in the field of apoptosis that relativeamounts of pro- and anti-survival Bcl-2 family of proteins determinewhether the cell will undergo cell death; if more Bcl-xL is present,then pores are non-permeable to pro-apoptotic molecules and the cellsurvives. Similar to Bcl-2, Bcl-xL has been implicated in the survivalof cancer cells by inhibiting the function of p53, a tumor suppressor.

The exosomes comprising Bcl-xL protein are prepared by expressing afusion protein composed of an exosome specific marker and a targetprotein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with Bcl-xL protein can be used forthe regulation of apoptosis.

Etc.

Multifunctional Signal Molecules: AIMP (Aminoacyl-tRNASynthase-Interacting Multifunctional Proteins)

Aminoacyl tRNA synthase complex-interacting multifunctional protein 1(AIMP1) is a non-catalytic component of the multi-synthase complex.Stimulates the catalytic activity of cytoplasmic arginyl-tRNA synthase.Possesses inflammatory cytokine activity. Negatively regulates TGF-betasignaling through stabilization of SMURF2 by binding to SMURF2 andinhibiting its SMAD7-mediated degradation. Involved in glucosehomeostasis through induction of glucagon secretion at low glucoselevels. Promotes dermal fibroblast proliferation and wound repair.

Plays a role in angiogenesis by inducing endothelial cell migration atlow concentrations and endothelial cell apoptosis at highconcentrations. Induces maturation of dendritic cells and monocyte celladhesion. Modulates endothelial cell responses by degrading HIF-1Athrough interaction with PSMA7.

Aminoacyl tRNA synthase complex-interacting multifunctional protein 2(AIMP2) is required for assembly and stability of the aminoacyl-tRNAsynthase complex. Mediates ubiquitination and degradation of FUBP1, atranscriptional activator of MYC, leading to MYC down-regulation whichis required for alveolar type II cell differentiation. Accumulates inbrains affected by autosomal-recessive juvenile Parkinsonism, idiopathicParkinson disease and diffuse Lewy body disease.

The exosomes comprising AIMP1 and AIMP2 proteins are prepared byexpressing a fusion protein composed of an exosome specific marker and atarget protein massively in the cell producing the exosomes at a highconcentration. The exosomes loaded with AIMP1 or AIMP2 protein can beused for multifunctional regulation.

Fluorescent Proteins (mCherry, GFP)

Fluorescent proteins are members of a structurally homologous class ofproteins that share the unique property of being self-sufficient to forma visible wavelength chromophore from a sequence of 3 amino acids withintheir own polypeptide sequence. It is common research practice forbiologists to introduce a gene (or a gene chimera) encoding anengineered fluorescent protein into living cells and subsequentlyvisualize the location and dynamics of the gene product usingfluorescence microscopy.

The most popular applications of fluorescent proteins involve exploitingthem for imaging of the localization and dynamics of specific organellesor recombinant proteins in live cells. For imaging of a specificorganelle, standard molecular biology techniques are used to fuse thegene encoding the fluorescent protein to a cDNA encoding a protein orpeptide known to localize to that specific organelle. This fusion isdone such that the chimeric gene will be expressed as a singlepolypeptide, creating a covalent link between the targeting motif andthe fluorescent protein. A plasmid containing the chimeric gene undercontrol of a suitable promoter is used to transfect mammalian cells thatthen express the gene to produce the corresponding chimeric protein. Thechimera localizes to the target organelle and thus renders itfluorescent. Through the use of fluorescence microscopy, the morphology,dynamics, and distribution of the organelle can be imaged as a functionof time.

mCherry is a monomeric fluorescent construct with peakexcitation/emission at 587 nm/610 nm, respectively. It is resistant tophotobleaching and is stable. It matures quickly, with a t0.5 of 15minutes, allowing it to be visualized soon after translation.

The green fluorescent protein (GFP) is a protein composed of 238 aminoacid residues (26.9 kDa) that exhibits bright green fluorescence whenexposed to light in the blue to ultraviolet range. Although many othermarine organisms have similar green fluorescent proteins, GFPtraditionally refers to the protein first isolated from the jellyfishAequorea Victoria. The GFP from A. Victoria has a major excitation peakat a wavelength of 395 nm and a minor one at 475 nm. Its emission peakis at 509 nm, which is in the lower green portion of the visiblespectrum.

The present invention prepared exosomes loaded with mCherry or GFPprotein and confirmed that mCherry or GFP protein was delivered to thecytosol of the target cells. The results indicate that the exosome ofthe present invention loaded with mCherry or GFP protein can be used forimaging of the localization and dynamics of exosomes and linked proteinsin live cells or animal.

Nucleic Acid-Binding Proteins (Ex. RNPs)

Nucleoproteins are any proteins that are structurally associated withnucleic acids, either DNA or RNA. A deoxyribonucleoprotein (DNP) is acomplex of DNA and protein. The prototypical examples are nucleosomes,complexes in which genomic DNA is wrapped around clusters of eighthistone proteins in eukaryotic cell nuclei to form chromatin. Protaminesreplace histones during spermatogenesis. DNPs in this kind of complexinteract to generate a multiprotein regulatory complex in which theintervening DNA is looped or wound. The DNPs participate in regulatingDNA replication and transcription.

A ribonucleoprotein (RNP) is a complex of RNA and protein. The enzymetelomerase, vault ribonucleoproteins, RNase P, hnRNP and small nuclearRNPs (snRNPs), and ribosomes are ribonucleoproteins. The RNPs play arole of protection. mRNAs never occur as free RNA molecules in the cell.They always associate with ribonucleoproteins and function asribonucleoprotein complexes.

The exosomes comprising DNPs or RNPs are prepared by expressing a fusionprotein composed of an exosome specific marker and a target proteinmassively in the cell producing the exosomes at a high concentration.The exosomes loaded with DNPs or RNPs can be used for geneticregulations or nucleic acid transportable exosomes.

The present invention confirmed that the cargo protein was successfullydelivered to cytosol of a target cell by using the exosome containingthe cargo protein therein, and thereby the present invention provides amethod to treat disease using exosome by regulating intracellularsignaling efficiently in cytosol.

Another object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating inflammatory diseases containingthe exosome as an active ingredient.

Another object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating cancer containing the exosome asan active ingredient.

Another object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating oxygen-related diseasescontaining the exosome as an active ingredient.

Another object of the present invention is to provide a composition forproducing a conditional knockout allele of a target gene containing theexosome as an active ingredient.

Another object of the present invention is to provide a DNA sequencemanipulating composition containing the exosome as an active ingredient.

Another object of the present invention is to provide a pharmaceuticalcomposition for preventing or treating Gaucher's disease containing theexosome as an active ingredient.

To develop an efficient method for preparing exosome containing a cargoprotein, the present inventors studied with various attempts. In thecourse of our study, the inventors paid attention on exosome specificmarkers (CD9, CD63, CD81, and CD82). These markers belong to thetetraspanin family and are commonly 4-times penetration type membraneproteins. The present inventors predicted that when a cargo protein wasconjugated on the membrane protein of exosome, the cargo protein wouldbe relatively easily included in the inside of exosome.

By expressing the fusion protein composed of an exosome specific markerwhich is rich especially on exosome membrane and can penetrate cellmembrane and a cargo protein in the cell producing exosome endogenouslyat a high concentration, exosome containing a cargo protein can bemassively produced.

Particularly, the method for preparing exosome comprising a cargoprotein of the present invention is characterized by introduction ofpolynucleotide encoding the fusion protein composed of an exosomespecific marker and a cargo protein in the cell producing exosome.

At this time, in the prepared exosome, a cargo protein is fused with anexosome specific marker embedded in exosome membrane.

The said cargo protein is bound to the membrane protein of exosome andis not separated even after it arrives at the target cell. To solve thisproblem, various attempts have been made. As a result, a technique hasbeen developed for the preparation of exosome comprising a cargo proteinby conjugating a cargo protein temporarily to a marker protein. Forexample, a photo-specific binding protein such as CIBN and CRY2 can beused herein. Particularly, CIBN is expressed in a form fused with CD9,which is one of the marker proteins. In the meantime, a gene encodingthe fusion protein of CRY2 and a cargo protein is introduced in the cellproducing exosome. The CIBN-CD9 fusion protein expressed in the exosomeproduction cell can be included due to CD9. At this time, when the cellis irradiated with blue LED light, CRY2 domain of the cargo protein-CRY2fusion protein expressed in the exosome production cell is bound to theCD9 fused CIBN domain. As a result, the reversible ‘cargoprotein-CRY2-CIBN-CD9 fusion protein’ is produced. This fusion proteincan be included in the inside of exosome due to CD9. Once exosomecontaining a cargo protein therein is produced and the irradiation withthe blue LED light is terminated, CIBN-CRY2 link is broken and therebythe cargo protein remains in exosome as being apart from the cellmembrane of exosome, resulting in the preparation of exosome comprisingthe cargo protein (FIGS. 5˜10).

This kind of exosome prepared by the method of the present invention iscompletely different in its effect from the conventional exosomecontaining a target material. The conventional exosome is expressed asbeing fused onto an exosome specific marker in order to present a cargoprotein inside of the exosome, so that the cargo protein, even though itis included in the inside of the exosome, it is not free and insteadpresented as being attached on the membrane of exosome, suggesting thatthe cargo protein cannot be separated from the membrane of exosome andtherefore it can be delivered into a target cell only when the exosomeis fused on the cell wall of the target cell. Moreover, even after thefusion onto the target cell, the cargo protein remains as beingconjugated to the membrane of exosome. Therefore, the probability thatthe cargo protein exhibits its effect in the target cell is very low.However, the exosome of the present invention presents a cargo proteinwhich resides as free and not being conjugated on the membrane ofexosome. So, when such exosome enters cytosol by endocytosis of thetarget cell, it does not adhere to the membrane of exosome, and when theexosome is decomposed therein, the included cargo protein can bedelivered in cytosol and is free to move in cytosol of the target cell,suggesting that the cargo protein is fully active with its physiologicalactivity in the target cell cytosol (FIG. 11).

The binding level of the cargo protein to the marker protein can bechanged according to the intensity of the light to be irradiated.Therefore, by regulating the intensity of the light, the concentrationof the cargo protein collected in exosome can be controlled.

The method for preparing exosome containing a cargo protein by using aphoto-specific binding protein has not been reported yet and wasproposed first by the present inventors.

Particularly, the method for preparing exosome containing a cargoprotein of the present invention is composed of the following steps: (a)introducing the polynucleopeptide encoding the fusion protein (fusionprotein I) composed of an exosome specific marker and the firstphoto-specific binding protein and the polynucleotide encoding thefusion protein (fusion protein II) composed of a cargo protein and thesecond photo-specific binding protein that can be linked to the firstphoto-specific binding protein in the exosome production cell; (b)irradiating the exosome production cell with light that can cause theconjugation between the first photo-specific binding protein and thesecond photo-specific binding protein; and (c) terminating theirradiation after the production of exosome finished in the exosomeproduction cell.

The term “exosome” in the present invention indicates a small vesiclewith the plasma membrane structure, which is originated from anintracellular specific compartment called multi-vesicular bodies (MVBs)and is released or secreted from the cell.

In this invention, exosome plays a role as a carrier to deliver a cargoprotein into a target cell or tissue by carrying the cargo protein initself. At this time, the cargo protein carried by the exosome works forthe target cell or tissue to help the treatment or diagnosis of aspecific disease.

The term “exosome production cell” in this invention indicates the cellthat is able to produce exosome.

In this invention, the exosome production cell is not limited but ispreferably exemplified by B-lymphocyte, T-lymphocyte, dendritic cell,megakaryocyte, macrophage, stem cell, and tumor cell, etc. For example,in this invention, HEK293T cell that is a kind of immortalized cell linewas used as the exosome production cell.

The term “exosome specific marker” in this invention indicates a proteinwhich is rich on the membrane of exosome.

In this invention, the exosome specific marker is not limited but ispreferably exemplified by CD9, CD63, CD81, and CD82, etc. For example,in a preferred embodiment of the present invention, CD9 was used as theexosome specific marker. CD9, CD63, CD81, and CD82 are 4-timespenetration type membrane proteins that allow the cargo protein to beeasily present in exosome when the cargo protein is bound to themembrane protein of the exosome.

The term “photo-specific binding protein” in this invention is alsocalled photo-induced heterodimer formation protein or photo-inducedhomodimer formation protein, which indicates a protein that is able toform a heterodimer by combining with different proteins or to form ahomodimer by combining with another protein in the same kind when thelight of a specific wavelength is irradiated.

In this invention, the photo-specific binding protein is not limited butis preferably exemplified by the photo-induced heterodimer formationprotein or CIB (cryptochrome-interacting basic-helix-loop-helixprotein), CIBN (N-terminal domain of CIB), PhyB (phytochrome B), PIF(phytochrome interacting factor), FKF1 (Flavinbinding, Kelch repeat,F-box 1), GIGANTEA, CRY (cryptochrome), and PHR (phytolyase homologousregion), etc.

In particular, when the photo-specific binding protein is thephoto-induced heterodimer formation protein, two types of photo-specificbinding protein (the first and the second photo-specific bindingproteins) can be used. When the first photo-specific binding protein isCIB or CIBN, the second photo-specific binding protein can be CRY orPHR. When the first photo-specific binding protein is PhyB, the secondphoto-specific binding protein can be PIF. When the first photo-specificbinding protein is GIGANTEA, the second photo-specific binding proteincan be FKF1.

For example, in a preferred embodiment of the present invention, CIBNwas used as the first photo-specific binding protein, and CRY2 was usedas the second photo-specific binding protein. The wavelength of thelight used herein was the blue light with 460˜490 nm. The intensity ofthe light was 20˜50 μW.

In the meantime, in order to confirm the expression and to find out thelocation of the first fusion protein composed of the exosome specificmarker and the first photo-specific binding protein expressed therein, amarker protein can be fused thereto. For example, in a preferredembodiment of the invention, the fluorescent protein EGFP was insertedin the first fusion protein wherein CIBN and CD9 or GIGANTEA and CD arelinked together. So, the expression pattern (expression and expressionlevel) and the intracellular location of the first fusion protein can beinvestigated by the expression of the first fusion protein as harboringthe fluorescent protein EGFP.

The term “cargo protein” in this invention indicates a protein which isexpressed as a fusion protein conjugated with the second photo-specificbinding protein to locate the cargo protein inside the exosome.

In this invention, the cargo protein can be carried by exosome afterbeing expressed in cells. The cargo protein is not limited but ispreferably a disease treating protein or disease diagnosing protein. Forexample, in a preferred embodiment of the present invention, mCherrywith fluorescence was used as the cargo protein.

An example of the cargo proteins in the present invention is selectedfrom, but not limited to, Matrix metalloproteinases (MMPs) proteins,Tissue inhibitor of metalloproteinases (TIMPs) proteins, caspasesproteins, caspases inhibitory proteins, cathepsins proteins or cathepsininhibitory proteins,

wherein,

MMPs proteins are such as, but not limited to, MMP1 protein (SEQ ID NO:13);

TIMPs proteins are such as, but not limited to, TIMP1 protein (SEQ IDNO: 14), TIMP2 protein (SEQ ID NO: 15), TIMP3 protein (SEQ ID NO: 16),or TIMP4 protein (SEQ ID NO: 17);

caspases proteins are such as, but not limited to, casepase 1 protein(SEQ ID NO: 18), casepase 2 protein (SEQ ID NO: 19), casepase 3 protein(SEQ ID NO: 20), casepase 4 protein (SEQ ID NO: 21), casepase 5 protein(SEQ ID NO: 22), casepase 6 protein (SEQ ID NO: 23), casepase 7 protein(SEQ ID NO: 24), casepase 8 protein (SEQ ID NO: 25), casepase 9 protein(SEQ ID NO: 26), casepase 10 protein (SEQ ID NO: 27), casepase 11protein (SEQ ID NO: 28), casepase 12 protein (SEQ ID NO: 29), casepase13 protein (SEQ ID NO: 30), or casepase 14 protein (SEQ ID NO: 31);

caspases inhibitory proteins are such as, but not limited to, proteinsinhibiting caspase proteins represented by SEQ ID NO: 18-31 or anyproteins inhibiting caspase;

cathepsins proteins are such as, but not limited to, cathepsins Aprotein (SEQ ID NO: 32), cathepsins B protein (SEQ ID NO: 33),cathepsins C protein (SEQ ID NO: 34), cathepsins D protein (SEQ ID NO:35), cathepsins E protein (SEQ ID NO: 36), cathepsins F protein (SEQ IDNO: 37), cathepsins G protein (SEQ ID NO: 38), cathepsins H protein (SEQID NO: 39), cathepsins K protein (SEQ ID NO: 40), cathepsins L1 protein(SEQ ID NO: 41), cathepsins L2 protein (SEQ ID NO: 42), cathepsins 0protein (SEQ ID NO: 43), cathepsins S protein (SEQ ID NO: 44),cathepsins W protein (SEQ ID NO: 45), or cathepsins Z protein (SEQ IDNO: 46); and

cathepsin inhibitory proteins are such as, but not limited to, proteinsinhibiting cathepsin proteins represented by SEQ ID NO: 32-46 or anyprotein inhibiting cathepsins.

Another example of the cargo proteins in the present invention isselected from, but not limited to, Cre recombinase, Cas protein,Caspase-activated DNase (CAD) proteins, β-glucocerebrosidase (GBA), p38mitogen-activated protein kinases, Phosphatase and tensin homolog(PTEN), Janus kinase (JAK), ubiquitin ligase, luciferase, peroxiredoxin(Prx) I or II, protein inhibiting NF-κB, MyoD proteins, Tbx18 proteins,p53 proteins, High mobility group box 1 (HMGB1) proteins, neurogenicdifferentiation1 (Neuro-D1) proteins, Interferon regulatory factor 5(IRF5) proteins, Interferon regulatory factor 3 (IRF3) proteins, Signaltransducer and activator of transcription 1 (STAT1) proteins, Suppressorof cytokine signaling 3 (SOCS3) proteins, Signal transducer andactivator of transcription 2 (STAT2) proteins, proteins inhibitingphosphorylated STAT3 (pYSTAT3), Bax (Bcl2-associated X protein), B-celllymphoma-extra-large (Bcl-xX) proteins, Aminoacyl-tRNAsynthase-interacting multifunctional proteins (AIMPs), mCherry proteins,green fluorescent proteins (GFP), or nucleoproteins binding to nucleicacid,

wherein,

Cre recombinase recombines the DNA between loxP sites by recognizingthem in DNA and includes, but not limited, Cre recombinase representedby SEQ ID NO: 9;

Cas protein has endonuclease or nickase activity when it combines thecomplex with guide RNA. In some embodiment, Cas protein is Cas9 proteinsuch as Cas protein represented by SEQ ID NO: 10, its mutant, or Cpf1protein such as amino acids represented by SEQ ID NO: 11;

CAD protein is such as the amino acids represented by SEQ ID NO: 47;

β-glucocerebrosidase (GBA) is such as the amino acids represented by SEQID NO:12;

p38 mitogen-activated protein kinases (p38 MAPKs) proteins are such asp38-α or its mutants and include amino acids represented by SEQ ID NOs:48-51;

Inhibitor kappa B kinase (IKK) proteins are such as the amino acidsrepresented by SEQ ID NO: 83;

Nuclear factor-kappa B (NF-κB) proteins are such as the amino acidsrepresented by SEQ ID NO: 84;

Phosphatase and tensin homolog (PTEN) proteins are such as the aminoacids represented by SEQ ID NO: 52;

Janus kinase (JAK) proteins include JAK1, JAK2, JAK3 and TYK2, whereinJAK1 proteins are such as the amino acids represented by SEQ ID NO: 53,JAK2 proteins are such as the amino acids represented by SEQ ID NO: 54,JAK3 proteins are such as the amino acids represented by SEQ ID NO: 55,and TYK2 proteins are such as the amino acids represented by SEQ ID NO:56; ubiquitin ligase proteins include c-CBL, PRKN, RBX1, TRAF2 and Mdm2,wherein ubiquitin ligase proteins are such as the amino acidsrepresented by SEQ ID NO: 57 to 61;

luciferase proteins are such as the amino acids represented by SEQ IDNO: 62;

peroxiredoxin (Prx) I or II has the effect of inhibiting cytotoxicityfrom oxidative stress, wherein peroxiredoxin I is such as the aminoacids represented by SEQ ID NO: 7, and peroxiredoxin II is the aminoacids represented by SEQ ID NO: 8;

protein inhibiting NF-κB is super-repressor-IκB which inactivates NF-κBby binding with it in cytoplasm, wherein the super-repressor-IκBprotein, which is S32A and S36A mutant form of IκB, is notphosphorylated by IκB Kinase (IKK) and consequently it can continuouslyinhibit NF-κB, and NF-κB inhibiting proteins are such as the amino acidsrepresented by one of SEQ ID NO: 1 to 5, exemplified by IκB-α, IκB-β,IκB-ε, BCL-3 or their mutant;

MyoD proteins are such as the amino acids represented by SEQ ID NO: 63;

Tbx18 proteins are such as the amino acids represented by SEQ ID NO: 64;

p53 proteins are such as the amino acids represented by SEQ ID NO: 65;

High mobility group box 1 protein (HMGB1) proteins are such as the aminoacids represented by SEQ ID NO: 66;

Neurogenic differentiation1 (Neuro-D1) proteins are such as the aminoacids represented by SEQ ID NO: 67;

Interferon regulatory factor 5 (IRF5) proteins are such as the aminoacids represented by SEQ ID NO: 68;

Interferon regulatory factor 3 (IRF3) proteins are such as the aminoacids represented by SEQ ID NO: 69;

Signal transducer and activator of transcription 1 (STAT1) proteins aresuch as the amino acids represented by SEQ ID NO: 70;

Suppressor of cytokine signaling 3 (SOCS3) proteins are such as theamino acids represented by SEQ ID NO: 71;

Signal transducer and activator of transcription 2 (STAT2) proteins aresuch as the amino acids represented by SEQ ID NO: 72;

proteins inhibiting phosphorylated STAT3 (pySTAT3) including pySTAT3intrabody antibody proteins, which binds to pySTAT3 to deactivatepySTAT3, and are such as the amino acids represented by SEQ ID NO: 73 orany proteins inhibiting pySTAT3;

Bax (Bcl2-associated X protein) is such as the amino acids representedby SEQ ID NO: 6;

B-cell lymphoma-extra-large (Bcl-xL) proteins are such as the aminoacids represented by SEQ ID NO: 74;

Aminoacyl-tRNA synthase-interacting multifunctional proteins (AIMPs)include AIMP1 and AIMP2, wherein AIMP1 proteins are such as the aminoacids represented by SEQ ID NO: 75 and AIMP2 proteins are such as theamino acid represented by SEQ ID NO: 76;

mCherry proteins are such as the amino acids represented by SEQ ID NO:77;

green fluorescent protein (GFP) are such as the amino acids representedby SEQ ID NO: 78;

nucleoproteins binding to nucleic acids include deoxyribonucleoprotein(DNP) binding to DNA or ribonucleoprotein (RNP) binding to RNA, whereinDNP includes RBBP4 or NAP1L4 and RNP include Telomerase, Heterogenousnuclear ribonucleoprotein K (HNRNPK) and wherein nucleoproteines aresuch as the amino acids represented by SEQ ID NOs: 79-82, nucleosome,protamine, small nuclear RNPs (snRNPs) or mutants thereof, or anyproteins binding to nucleic acid.

The term “culture” in this invention indicates a method to grow cells ormicroorganisms in a properly controlled environment.

In this invention, a transformant was cultured for 1˜3 days and then themedium was replaced with a serum-free medium, followed by furtherculture for 2˜5 days.

In this invention, the method for culturing the transformant is any ofthose well known to those in the art.

The said medium herein indicates a notified medium widely used foranimal cell culture, which can be selected from the group consisting ofcommercially available serum-free media, protein-free media, andchemically defined media.

The serum-free media above are used for animal cell culture, which arefree from bovine serum and are exemplified by SFM4CHO (HyClone) andEX-Cell (JHR Bioscience). Insulin like growth factor I (IGF-I),ethanolamine, ferric chloride, and phosphatidyl choline can be added tothe media, but not always limited thereto.

The protein-free media above are animal cell culture media, from whichanimal originated proteins especially high molecular proteins inparticular having the molecular weight of at least 10 kDa areeliminated. The protein-free media can be ProCHO (Lonza) and PF-CHO(HyClone), but not always limited thereto.

The chemically defined media above are animal cell culture media whichdo not include any animal originated components and instead havecomponents all having defined chemical structures. The chemical definedmedia can be CDM4CHO (HyClone), PowerCHO2CD (Lonza), and CD-optiCHO(Life Technologies), but not always limited thereto.

The term “the first fusion protein” in this invention indicates thefusion protein made by binding between the exosome specific marker andthe first photo-specific binding protein.

In this invention, the order of arrangement of the exosome specificmarker and the first photo-specific binding protein contained in thefirst fusion protein is not limited as long as the first photo-specificbinding protein is located in the direction toward the inside of exosomewhen the first fusion protein is expressed in the exosome productioncell. For example, N-terminal of the first photo-specific bindingprotein can be conjugated to C-terminal of the exosome specific marker.

The exosome specific marker and the first photo-specific binding proteinwhich compose the first fusion protein are linked directly each other orcan be connected by a linker. The linker above is not limited as long asthe first fusion protein is expressed in the exosome production cellwith presenting the first photo-specific binding protein located in thedirection toward the inside of exosome, but is preferably a peptidelinker composed of amino acids and more preferably a flexible peptidelinker. The peptide linker can be expressed by using an expressionvector wherein the nucleic acids encoding the linker are connected withother nucleic acids encoding each domain in frame.

The term “the second fusion protein” indicates a fusion protein in whichthe second photo-specific binding protein and the cargo protein arecombined.

In this invention, the order of arrangement of the second photo-specificbinding protein and the cargo protein contained in the second fusionprotein is not limited as long as the second fusion protein is locatedinside of exosome as being conjugated with the first photo-specificbinding protein region of the first fusion protein in the exosomeproduction cell. For example, N-terminal of the cargo protein can beconjugated to C-terminal of the second photo-specific binding protein.

The second photo-specific binding protein and the cargo protein whichcompose the second fusion protein are linked directly each other or canbe connected by a linker. The linker above is not limited as long as thesecond fusion protein is located inside of exosome as being conjugatedwith the first photo-specific binding protein of the first fusionprotein in the exosome production cell, but is preferably a peptidelinker composed of amino acids and more preferably a flexible peptidelinker. The peptide linker can be expressed by using an expressionvector wherein the nucleic acids encoding the linker are connected withother nucleic acids encoding each domain in frame.

In addition, each fusion protein above can include a polypeptide havingthe sequence wherein at least one amino acid residues are different fromthose in the wild type amino acid sequence of each domain includedtherein. Amino acid exchange in proteins and polypeptides withoutchanging the overall activity of a molecule is well known to those inthe art. The most common exchange occurs between Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. Inaddition, a protein having increased structural stability against heator pH or increased protein activity due to mutation or modification ofamino acid sequence can be included.

Lastly, the fusion protein above or the polypeptide of each domaincomprising the fusion protein can be prepared by the chemical peptidesynthesis method well informed to those in the art, or prepared by thefollowing method. A gene encoding each domain is amplified by PCR(polymerase chain reaction) or synthesized by the conventional methodwell known to those in the art. The gene is cloned in an expressionvector and expressed.

In the meantime, each fusion protein can be expressed in the exosomeproduction cell by introducing a polynucleotide encoding each fusionprotein in the exosome production cell. At this time, the polynucleotideis introduced in the exosome production cell by the conventional methodwell informed to those in the art. For example, an expression vector canbe used for the introduction.

The term “expression vector” in this invention is a recombinant vectorcapable of expressing a target peptide in host cells. This vectorindicates a gene construct containing essential regulators operablylinked so as to express the gene insert. The expression vector includesexpression control elements such as a start codon, a termination codon,a promoter, and an operator. The start codon and termination codon aregenerally understood as a part of the nucleotide sequence encoding apolypeptide. They are supposed to be working when a gene construct isintroduced and to reside in a coding sequence in frame. The promoter ofthe vector can be constitutive or inductive.

The term “operably linked” in this invention indicates the status whenthe nucleic acid expression regulation sequence functioning as usual andthe nucleic acid sequence encoding a cargo protein or RNA are linked byfunctional linkage. For example, a promoter is operably linked to anucleic acid sequence encoding a protein or RNA in order to affect theexpression of the coding sequence. The functional linkage with anexpression vector can be achieved by the recombinant DNA technology wellknown to those in the art, and particularly the site-specific DNAcleavage and linkage can be achieved by using the conventional enzymewell known to those in the art.

The said expression vector can include a signal sequence for thedischarge of a fusion polypeptide in order to promote the separation ofa protein from the cell culture medium. A specific initiation signalmight be necessary for the efficient translation of the inserted nucleicacid sequence. These signals contain ATG start codon and its neighboringsequences. In some cases, an exogenous translational control signal,which may include the ATG start codon, should be provided. Theseexogenous translational control signals and start codon can be variousnatural and synthetic sources. The expression efficiency can beincreased by the introduction of appropriate transcription ortranslation enhancers.

In a preferred embodiment of the present invention, the expressionvector is able to express a cargo protein conjugated with a tag in orderto confirm the insertion of a cargo protein inside the exosome. The tagherein is to confirm the presence of a cargo protein, which can beconjugated to the region opposite to the region of the secondphoto-specific binding protein conjugation. For example, a fluorescentprotein such as a red fluorescent protein and a green fluorescentprotein is used as a tag to be conjugated to C-terminal of a cargoprotein.

The cargo protein prepared as described above is expressed in theexosome production cell. Once exosome is produced, it is investigatedwhether or not the fluorescent protein tag is detected, by which thepresence of the cargo protein in exosome can be confirmed.

The term “light” in this invention indicates the light to be irradiatedin order to combine temporarily the first photo-specific binding proteinand the second photo-specific binding protein expressed in the exosomeproduction cell.

As described hereinbefore, the first photo-specific binding protein isexpressed as the first fusion protein conjugated with the exosomespecific marker, while the second photo-specific binding protein isexpressed as the second fusion protein conjugated with the cargoprotein. When the light is irradiated to the exosome production cell,the first photo-specific binding protein is combined with the secondphoto-specific binding protein, and as a result the fusion proteincomplex comprising the exosome specific marker—the first photo-specificbinding protein—the second photo-specific binding protein—the cargoprotein is formed temporarily. When exosome is produced in the exosomeproduction cell, the cargo protein can be linked to the exosome due tothe exosome specific marker. At this time, the cargo protein presentsinside the exosome and when the irradiation with the light is stoppedafter the production of the exosome, the first photo-specific bindingprotein is separated from the second photo-specific binding protein andthereby the cargo protein included in the exosome is to be dischargedtogether with the exosome as being a part of the exosome. It ispreferred for the light to be irradiated to the cell intermittentlyrather than continually in order to deliver the cargo protein inside theexosome more efficiently. That is, when the light is irradiatedintermittently, the conjugation and separation of the firstphoto-specific binding protein and the second photo-specific bindingprotein repeat so that the probability that the cargo protein isintroduced into the exosome can be increased.

In the meantime, the wavelength of the light enough to induce thebinding of the first photo-specific binding protein with the secondphoto-specific binding protein varies from the kinds of the first andthe second photo-specific binding proteins. The wavelength of the lightthat induces the binding of the first photo-specific binding protein andthe second photo-specific binding protein depends on the type of theproteins. So, the proper wavelength of the light can be selected asknown to those in the art. For example, in order to link CRY2 to CIBN,the light with the wavelength of 460˜490 nm is preferred. If the lightis irradiated less than 10 minutes, CRY2 and CIBN are separated fromeach other. When PhyB is combined with PIF, the light with thewavelength of 650 nm is irradiated for 10 minutes. When the light withthe wavelength of 750 nm is irradiated for 5 minutes, PhyB and PIF areseparated from each other. When FKF1 is combined with GIGANTEA, thelight with the wavelength of 460 nm is irradiated for 30 minutes. In apreferred embodiment of the present invention, in order to induce thebinding of CIBN and CRY2, the light with the wavelength of 460˜490 nmwas irradiated.

In a preferred embodiment of the present invention, the CRY2/mCherryfusion protein and the CIB/CD9 fusion protein were expressed in HEK293T,the immortalized cell line producing a large amount of exosome. As aresult, the distribution of mCherry protein uniformly distributed incytosol was found to be in cell membrane and endosome-like structuremembrane when the blue light was irradiated (FIG. 7). Similar resultswere observed when the FKF1/mCherry fusion protein and the GIGANTEA/CD9fusion protein were expressed in HEK293T cells (FIG. 12). TheCRY2/mCherry fusion protein and the CIBN/CD9 fusion protein wereexpressed in HEK293T cells, followed by irradiation with the blue lightwith regulating the intensity of the light. As a result, when the lightwas irradiated with the intensity of 20˜50 μW, the level of mCherryprotein collected in exosome was the highest (FIG. 9). The exosomesisolated from the cells were treated to HT1080 cells at theconcentration of approximately 250 μg/ml. As a result, the exosomes didnot show any specific cytotoxicity against the HT1080 cells and it wasconfirmed that the mCherry protein was delivered in the cytosol thereof(FIG. 10).

To compare the efficiency of introducing the cargo protein in exosomeand the efficiency of exosome transfer to the target cell with those ofthe conventional methods, XPACK vector was used for the conventionalmethod and the expression vectors of the CRY2/mCherry fusion protein andthe CIBN/CD9 fusion protein were introduced in HEK293T cells. Then, theproduction of the cargo protein in exosome was compared. As a result, itwas confirmed that the introduction efficiency was remarkably high whenthe method of the present invention was used (FIG. 15). The exosomeseparated from the exosome production cell was treated to the targetcell (HeLa) to compare the expression of the cargo protein. When theexosome separated by the method of the present invention was used, theexpression of the cargo protein was the highest in the target cell (FIG.16).

In another preferred embodiment of the present invention, the presentinvention provides a vector for the production of exosome comprising (a)the first expression vector containing the polynucleotide encoding thefusion protein of the exosome specific marker and the firstphoto-specific binding protein (the first fusion protein); and (b) thesecond expression vector containing the multicloning site to which thepolynucleotide encoding the cargo protein can be introduced and thepolynucleotide encoding the second photo-specific binding protein to belinked to the first photo-specific binding protein above.

In the vector for the production of exosome provided by the presentinvention, the exosome specific marker, the first photo-specific bindingprotein, the exosome production cell, and the second photo-specificbinding protein are same as described above.

The term “transformed cells for exosome production” in this inventionindicates the cells capable of producing exosome by expressing the firstfusion protein wherein the polynucleotide encoding the fusion protein(the first fusion protein) of the exosome specific marker and the firstphoto-specific binding protein is introduced.

In this invention, the second expression vector includes apolynucleotide encoding the second photo-specific binding protein and aneighboring multicloning site. When a polynucleotide encoding a cargoprotein is inserted in the multicloning site, it is expressed as thefusion protein comprising the second photo-specific binding protein andthe cargo protein (the second fusion protein).

The vector for preparing exosome provided by the present invention cancontain one or more kinds of constituents, solutions, or devices usablenot only for the transformed cells for exosome production and theexpression vector; but also for the introduction of the expressionvector; for the culture of the transformed cells for exosome production;and for the separation and purification of the exosome produced from thetransformed cells for exosome production. For example, a buffer properfor the introduction of the expression vector and a medium and a vesselnecessary for the culture of the transformed cells for exosomeproduction can be additionally included.

The term “Cas protein” in this invention indicates the essential proteinin CRISPR/Cas system which form active endonuclease or nickase when Casprotein form the complex with two RNA called CRSPR RNA (crRNA) andtrans-activating crRNA (tracrRNA).

The term “guide RNA” in this invention indicates target DNA-specific RNAwhich is able to form complex with Cas protein and guides Cas protein totarget DNA.

In this invention, aforementioned guide RNA is able to be made by twoRNA, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) orsingle-chain RNA (sgRNA) by fusing the essential parts of crRNA andtracrRNA.

Aforementioned guide RNA is able to be dual RNA including crRNA andtracrRNA. If aforementioned RNA includes the essential parts and targetcomplementary parts of crRNA and tracrRNA, any guide RNA is being ableto be applied in this invention. Aforementioned crRNA is able tohybridize target DNA.

Aforementioned guide RNA is able to include one or more additionalnucleotide on 5′ terminal of single-chain guide RNA or crRNA in dualRNA.

Desirably, aforementioned guide RNA is able to include two additionalguanine nucleotides on 5′ terminal of single-chain guide RNA or crRNA indual RNA. Guide RNA is able to be delivered to cell or organism as RNAor guide RNA coding DNA. Guide RNA is able to be separated RNA, RNAincluded in virus vector, or coded in vector. Desirably, aforementionedvector is not limited but is able to be virus vector, plasmid vector, oragrobacterium vector.

Guide RNA coding DNA is able to be vector including guide RNA coding DNAsequence. For example, guide RNA is able to be delivered to cell ororganism by transfecting plasmid DNA that includes isolated guide RNA orguide RNA coding sequence and promoter. By other method, guide RNA canbe delivered to cell or organism by using virus-mediated gene delivery.

When guide RNA is transfected into cell or organism as isolated RNA, itcan be manufactured by in vitro transcription by using any in vitrotranscription systems known in industry. Desirably, guide RNA isdelivered to cell as isolated RNA rather than plasmid includingguide-RNA coding sequence. The term “isolated RNA” can be replaced by“naked RNA” in this invention. It is able to save cost and time in thatisolated RNA does not need cloning process. However, the usage ofplasmid DNA or virus-mediated gene delivery for guide RNA transfectionis not excluded.

The present invention provides the exosome prepared by the method of theinvention in which a cargo protein is included.

In another aspect, the present invention provides an exosome produced bythe above method, wherein the Cre recombinase is contained in theexosome.

In another aspect, the present invention provides an exosome prepared bythe above method, wherein the Cas9 protein is contained therein.

In another aspect, the present invention provides an exosome produced bythe above method, wherein GBA (β-glucocerebrosidase) protein iscontained therein.

In another aspect, the present invention provides an exosome produced bythe above method, wherein the peroxiredoxin (Prx) I or II protein iscontained therein.

In another aspect, the present invention provides an exosome produced bythe above method and comprising a protein that inhibits NF-kB.

In another aspect, the present invention provides an exosome prepared bythe above method, wherein Bax (Bcl-2-associated X protein) protein iscontained therein.

The exosome prepared by the method above contains a fusion protein (thefirst fusion protein) composed of an exosome specific marker and thefirst photo-specific binding protein on the plasma membrane thereof andanother fusion protein (the second fusion protein) composed of thesecond photo-specific binding protein that can be conjugated to thefirst photo-specific binding protein and a cargo protein. So, when suchexosome is treated to the target tissue cells, the second fusion proteinincluded in the exosome can be delivered to cytosol of the target tissuecells through the fusion of the plasma membrane.

The said exosome containing a cargo protein can be used for thetreatment of various diseases in vivo. For example, exosome containing aprotein polymer (for example, antibody, etc.) showing the anticanceractivity as a cargo protein is prepared, which is then treated to cancercells. That is, the exosome can be used as a biocompatible anticanceragent better acting than the conventional liposome.

This invention also provides the pharmaceutical components forinflammatory disease prevention and therapy including exosomes withNF-κB inhibiting protein.

Aforementioned inflammatory diseases are not limited but is preferablyexemplified by allergy, dermatitis, atopy, conjunctivitis,periodontitis, rhinitis, otitis media, laryngopharyngitis, tonsillitis,pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout,ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriaticarthritis, osteoarthritis, rheumatoid arthritis, periarthritis ofshoulder, tendonitis, tenosynovitis, peritendinitis, myositis,hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis,acute and chronic inflammatory diseases, sepsis, and ulcerative colitis,etc.

In the experimental examples in this invention, the present inventorsconfirmed that transfer of NF-κB activated by TNF-α to nucleus isinhibited by pretreating super-repressor-IκB: EXPLOR to HeLa cell toverify inflammation inhibitory effect mediated by TNF-α (FIG. 43. Left).In addition, inhibition of DNA binding of NF-κB activated by TNF-α wasconfirmed (FIG. 43. Right). Also, the present inventors confirmed thatsymptom of arthritis is decreased in mouse model which is inducedarthritis by collagen through injecting retro-orbital three times toverify inflammation inhibitory effects, and therebysuper-repressor-IκB:EXPLOR in this invention can be used as thepharmaceutical components for inflammatory disease prevention andtherapy.

This invention also provides the pharmaceutical components for cancerprevention and therapy including exosomes with Bax (Bcl-2 associated Xprotein).

Aforementioned cancer is not limited but is preferably exemplified bybreast cancer, colon cancer, lung cancer, small-cell lung cancer,gastric cancer, liver cancer, blood cancer, bone cancer, pancreaticcancer, skin cancer, head or neck cancer, skin or choroidal melanoma,eye cancer, peritoneal cancer, uterine cancer, ovarian cancer, rectalcancer, anal cancer, and cervical cancer, etc.

In the experimental examples in this invention, the present inventorsconfirmed that cytochrome c release is increased by pretreatingBax::EXPLOR to HeLa cell, and thereby Bax: EXPLOR can be used as thepharmaceutical components for cancer prevention and therapy.

This invention also provides the pharmaceutical components foranti-oxidation including exosomes with peroxiredoxin (Prx) I or II.

Also, this invention provides the pharmaceutical components forprevention and therapy of reactive oxygen species disease exemplified bycancer, arteriosclerosis, respiratory disease, osteoporosis, obesity,and degenerative dementia including exosomes with peroxiredoxin (Prx) Ior II.

Also, this invention provides the cosmetic ingredients foranti-oxidation including exosomes with peroxiredoxin (Prx) I or II.

Also, this invention provides the cosmetic ingredients for anti-aging ofskin including exosomes with peroxiredoxin (Prx) I or II.

In the experimental examples in this invention, the present inventorsconfirmed that cytotoxicity by oxidative stress is inhibitedstatistically significant by pretreating Prx I/II::EXPLOR to HeLa cellto verify the inhibitory effect on cytotoxicity by oxidative stressinduced by H₂O₂, and thereby Prx I/II::EXPLOR can be used as thepharmaceutical components for anti-oxidation or prevention and therapyof reactive oxygen species, or the cosmetic ingredients foranti-oxidation or anti-aging of skin.

This invention also provides the components for creating conditionalknockout allele of target gene including exosomes with Cre recombinase.

In the experimental examples of this invention, the present inventorsconfirmed the expression of ZsGreen reporter protein in Cre::EXPLORtreated HT1080 cell and HeLa cell, with identical results of pCMV-Crevector transfection as positive control, through detecting ZsGreenreporter expression after transfecting pCAG-loxP-STOP-loxP-ZsGreenencoded DNA into HT1080 and HeLa cell to verify the effect of Crerecombinase (FIGS. 19A and 19B). In addition, the present inventors wasable to confirm the ZsGreen expression on Cre::EXPOR treated primarymouse embryo neuron performed by experiment identical to aforementioned(FIG. 20). Also, the present inventors confirmed that EYFP is expressedon Cre::EXPLOR treated group after ventrolateral injection ofCre::EXPLOR on pCAG-lowP-STOP-loxP-eNpHR3.0-EYFP transgenic mouse toverify Cre-EXPLOR function in vivo (FIG. 21). Furthermore, it wasconfirmed that Cre::EXPLOR mainly targets neuron in mouse brain throughmerged neuronal region in the results of immunohistochemistry to verifyCre::EXPLOR targeting cell (FIG. 22), and thereby Cre::EXPLOR can beused as the components for creating conditional knockout allele oftarget gene.

This invention also provides the components for engineering DNA sequenceincluding exosomes with Cas9 protein and target DNA specific guide RNA(gRNA).

Aforementioned components are not limited but preferably inducesmutation on normal sequence or proofreads mutation. Mutation can benaturally occurred mutation or induced by pathogenic microbes. In otherword, mutation is occurred by infection of pathogenic microbes whenpathogenic microbes are detected and it becomes clear that biologicalsample is infected. Pathogenic microbe is not limited but it can bevirus or bacteria.

In the experimental examples in this invention, the present inventorsconfirmed exosome comprising a CRISPR/Cas9 protein can be prepared witha high yield.

This invention also provides the pharmaceutical components for curingGaucher disease including exosomes with β-glucocerebrosidase (GBA).

In the experimental examples in this invention, the present inventorsconfirmed that activity of β-glucocerebrosidase (GBA) is recovered bytreating GBA::EXPLOR to cells from Gaucher disease patients (FIG. 30),and thereby GBA::EXPLOR can be used as the pharmaceutical components forcuring Gaucher disease.

According to the method for preparing exosome containing a cargo proteinof the invention, exosome comprising a cargo protein can be preparedwith a high yield. Also, a cargo protein presents as being separatedfrom the membrane of exosome, so that it can be widely applied to treatdisease.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples. However, it will beappreciated that those skilled in the art, on consideration of thisdisclosure, may make modifications and improvements within the spiritand scope of the present invention.

Example 1: Preparation of Exosome

<1-1> Confirmation of the Binding of CIBN and CRY2 for the Production ofExosome

PcDNA3.1 (+) vector containing CIBN-EGFP-CD9 gene and pcDNA3.1 (+)vector containing mCherry-CRY2 gene were introduced into HEK293T cells,the exosome production cells, under the light-free condition, followedby culture for 24 hours. The medium was replaced with a serum-freemedium, followed by additional culture for 48 hours. Upon completion ofthe culture, the cells were irradiated with the blue light with thewavelength of 460˜490 nm. The location of red fluorescence shown inmCherry before and after the blue light irradiation was confirmed byusing a confocal microscope (FIG. 7).

FIG. 7 is a fluorescence image illustrating the changes in theintracellular location of mCherry protein according to the blue lightirradiation in the transformed HEK293T cells introduced withCIBN-EGFP-CD9 gene and mCherry-CRY2 gene. As shown in FIG. 7, before theblue light irradiation that could cause the binding of thephoto-specific binding proteins CIBN and CRY2, mCherry protein wasevenly distributed in the cytosol. However, after the blue lightirradiation, mCherry protein was concentrated in the membrane. Thisclustering of mCherry protein was analyzed to be caused by the bindingof CIBN and CRY2, the photo-specific binding proteins.

<1-2> Confirmation of the Binding of GIGANTEA and FKF1 for theProduction of Exosome

PcDNA3.1 (+) vector containing GIGANTEA-EGFP-CD9 gene and pcDNA3.1 (+)vector containing mCherry-FKF1LOV gene were used in this example. Theintracellular Exosome was confirmed by the same manner as described inExample <1-1>. (LOV in the FKF1LOV above is an abbreviation oflight-oxygen-voltage domain, which indicates the domain that binds toother proteins by light in FKF1 protein, so FKF1 and FKF1LOV are in factthe same herein).

Like Example <1-1>, as shown in FIG. 12, mCherry protein was evenlydistributed in the cytosol before the blue light irradiation that couldcause the binding of the photo-specific binding proteins GIGANTEA andFKF1. However, after the blue light irradiation, mCherry protein wasconcentrated in the membrane. This clustering of mCherry protein wasanalyzed to be caused by the binding of GIGANTEA and FKF1, thephoto-specific binding proteins.

Example 2: Exosome Production and the Effect of Light Intensity onExosome Production

Each expression vector respectively containing CIBN-EGFP-CD9 gene andmCherry-CRY2 gene was introduced into HEK293T cells under the LED lightwith the wavelength of 460 nm at the intensity of 0, 5, 20, 50, and 200μW, followed by culture for 24 hours. Then, the medium was replaced witha serum-free medium, followed by additional culture for 48 hours. Uponcompletion of the culture, the culture medium was separated, which wascentrifuged (3000×g, 15 minutes) to obtain the supernatant excludingcell debris. ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) was added to the obtainedsupernatant at the volume of 5 times the supernatant. After the mixing,centrifugation was performed (1500×g, 30 minutes) to obtain theprecipitated exosome. The obtained exosome was suspended in PBS,resulting in the exosome suspension. The exosome suspension was filteredwith a 0.2 μm filter using a syringe equipped with a 27-G needle. As aresult, exosome in the single size was obtained (FIG. 8). Then, exosomelysate was prepared by using lysis buffer, followed by immune-blottingto compare the amount of mCherry protein in the exosome (FIG. 9).

FIG. 9 is an immunoblot analysis image showing the results of measuringthe changes of the content of a cargo protein (mCherry protein) capturedin exosome according to the intensity of blue light. As shown in FIG. 9,when the cells were irradiated with blue light at the intensity of 20˜50μW, the amount of mCherry, the cargo protein, in exo some was thehighest. From the above results, it was confirmed that the content ofthe cargo protein captured in exosome could be regulated by controllingthe intensity of the light irradiated to the cells in the course of thebinding of the photo-specific binding proteins.

Example 3: Effect of Exosome Treatment

Each expression vector respectively containing CIBN-EGFP-CD9 gene andmCherry-CRY2 gene was introduced into HEK293T cells under the LED lightwith the wavelength of 460 nm at the intensity of 50 μW, followed byextracting exosome by the same manner as described in Example 2. Theextracted exosome was treated to HT1080 cells at the concentration of250 μg/ml for 24 hours. The HT1080 cells were fixed on 10% gelatin gelby adding with 0.1 M phosphate buffer (pH 7.4) containing 4% PFA and0.01% GA. The cells attached on the gelatin gel were cooled for a day byusing liquid nitrogen. Thin sections cut in 45 nm by usingcryoultramicrotome were obtained at −120° C. The thin sections wereimmuno-stained by using anti-mCherry antibody and Protein A-gold.MCherry protein was observed with Tecnai G2 Spirit Twin TEM (FIG. 10).

FIG. 10 is an electron micrograph illustrating the results ofinvestigation of the introduction of a cargo protein in target cellsafter treating the target cells (HT1080) with exosome containing thecargo protein (mCherry), wherein the left indicates the target cellsnot-treated with exosome and the right indicates the target cellstreated with exosome. As shown in FIG. 10, it was confirmed that thecargo protein was transferred into the target cells when the targetcells were treated with the exosome of the present invention.

Example 4: Analysis of Exosome with Cargo Protein

Each expression vector respectively containing CIBN-EGFP-CD9 gene andmCherry-CRY2 gene was introduced into HEK293T cells under the LED lightwith the wavelength of 460 nm at the intensity of 50 μW, followed byextracting exosome by the same manner as described in Example 2. Theextracted exosome was treated to HT1080 cells at the concentration of250 μg/ml for 24 hours. Then, red fluorescence was confirmed in mCherryprotein under a fluorescent microscope and the ratio of dead cells wascompared between the cells treated with exosome and the cellsnot-treated with exosome by LDH cell death assay (FIG. 11).

FIG. 11 is a set of a fluorescence image (a) illustrating the results ofinvestigation of the introduction of a cargo protein in target cellsafter treating the target cells (HT1080) with exosome containing thecargo protein (mCherry); and a graph (b) illustrating the results ofcomparison of the ratio of apoptotic cells induced by the treatment ofexosome. As shown in FIG. 11, it was confirmed that apoptosis did notinduced by the treatment of exosome.

Example 5: Exosome Production and the Comparison of IntroductionEfficiency of a Cargo Protein into the Produced Exosome

<5-1> Confirmation of Exosome Production Efficiency

To compare the exosome production and the introduction efficiency of acargo protein into the exosome produced thereby according to the presentinvention with those of the conventional method, the expression of thecargo protein in the exosome production cells was investigated bymeasuring the luciferase activity therein.

According to the conventional method, XPACK-Luciferase-mCherry wasintroduced in HEK293T cells by using XPACK (Systems Biosciences), thecommercial vector designed for exosome loading technique (XP). On theother hand, according to the method of the invention,Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 were introduced in HEK293Tcells (EXPLOR). Then, the luciferase activity in both cells was measuredto compare the efficiency of the two methods. The luciferase activitywas measured according to the manufacturer's instructions (LuciferaseAssay Reagent, Promega). The standard curve of the results was plotted,and then the number of exosomes in the cells was quantitativelycalculated.

As shown in FIG. 14, it was confirmed that the method using thephoto-specific binding proteins CIBN and CRY2 of the present inventionwas significantly higher in the introduction efficiency into exosomethan the conventional method (XP) (FIG. 14).

<5-2> Expression of a Cargo Protein in the Produced Exosome

The cells of Example <5-1> were cultured for 72 hours, followed byextracting exosome (Exoquick-TC, Systems biosciences). The concentrationof the cargo protein included in the exosomes separated by theconventional method (XP) or the method of the present invention wascompared indirectly by measuring the luciferase activity therein. Asshown in FIG. 15, it was confirmed that the method of the presentinvention could produce exosome containing a remarkably large amount ofthe cargo protein than the conventional method (FIG. 15).

<5-3> Comparison of the Introduction Efficiency of the Cargo Protein

The introduction efficiency (E) of the cargo protein was calculated bythe mathematical formula below based on the luciferase activity measuredin Examples <5-1> and <5-2>.

E=measured value of luciferase activity in produced exosome/measuredvalue of luciferase activity in exosome production cell  [MathematicalFormula 1]

As shown in FIG. 15, it was confirmed that the exosome produced usingthe binding of CRY2 and CIBN of the present invention exhibited 4 to 120times higher efficiency than those of the other comparative groups (FIG.15).

Example 6: Comparison of Exosome Transfer Efficiency to Target Cells

To compare the exosome transfer efficiency, the target cells weretreated with exosome containing the cargo protein. Particularly, HeLacells were treated with 5×10⁹ exosomes for 24 hours, and then thefluorescence intensity expressed in the cells was measured. As shown inFIG. 16, it was confirmed that the fluorescence intensity in the exosomeof the present invention (EXPLOR) was remarkably high (FIG. 16).

Therefore, it was confirmed that the method using the exosome of thepresent invention could deliver the cargo protein to the target cellsmore efficiently.

Experimental Examples Experimental Example 1: MMPs (MatrixMetalloproteinases)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andMMP-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofMMPs within the exosome is evaluated.

For the massive production of MMPs-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and MMP-mCherry-CRY2 gene are established,and exosomes are isolated and purified by Tangential Flow Filtration(TFF) method from culture supernatant.

Functional analysis of MMPs-loaded exosomes is performed in targetcells:

Target cells are treated with the MMPs-loaded exosomes to evaluate thefunctional enzymatic activity.

Animal models are administered with the MMPs-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 2: TIMPs (Tissue Inhibitor of Metalloproteinase)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andTIMP-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofTIMPs within the exosome is evaluated.

For the massive production of TIMPs-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and TIMP-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of TIMPs-loaded exosomes is performed in targetcells:

Target cells are treated with the TIMPs-loaded exosomes to evaluate thefunctional enzymatic activity.

Animal models are administered with the TIMPs-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 3: Caspases

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andcaspase-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofcaspases within the exosome is evaluated.

For the massive production of caspases-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and caspase-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of caspases-loaded exosomes is performed in targetcells:

Target cells are treated with the caspases-loaded exosomes to evaluatethe functional enzymatic activity.

Animal models are administered with the caspases-loaded exosomes by i.p.or i.v. to show therapeutic effect.

Experimental Example 4: Cathepsins

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andcathepsin-mCherry-Cry2 at 488 nm wavelength blue light, and the loadingof cathepsins within the exosome is evaluated.

For the massive production of cathepsins-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and cathepsin-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of cathepsins-loaded exosomes is performed in targetcells: Target cells are treated with the cathepsins-loaded exosomes toshow the functional enzymatic activity.

Animal models are administered with the cathepsins-loaded exosomes byi.p. or i.v. to show therapeutic effect.

Experimental Example 5: Cre Recombinase

<5-1> Production of Cre Recombinase-Loaded Exosome (Cre::EXPLOR)

A. Confirmation of Cre Recombinase in Exosome

The present inventor confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and Cre-mCherry-Cry2 to verify exosome loadingof Cre-recombinase with amino acids recorded as SEQ ID NO: 9. Inparticular, HEK293T exosome producing cells were additionally cultured48 hrs in Dulbecco's modified Eagle's medium (DMEM) without fetal bovineserum (FBS), after 24 hrs culture with transfected pcDNA3.1 (+) vectorincluding CIBN-EGFP-CD9 gene and Cre-mCherry-CRY2 gene in non-lightcondition. After finish culture, position of red fluorescence frommCherry was investigated by confocal microscopy before and after theirradiation of 488 nm wavelength blue light. This experiment wasperformed more than five times.

According to the results, binding between Cre-mCherry-CRY2 (red) andCIBN-EGFP-CD9 was confirmed (FIG. 18) and thereby exosome loading of Crerecombinase was verified.

B. Production of Cre Recombinase-Loaded Exosome (Cre::EXPLOR)

The present inventors performed following experiment to yield the Crerecombinase-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and Cre-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. After 24 hrs culture, cells were changedtheir medium as it without fetal bovine serum (FBS) and additionallycultured during 48 hrs on 50 μW power of 488 nm wavelength blue light.After finishing culture, supernatant removed cell debris was yielded bycentrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and Cre-mCherry-CRY2 gene were cultured in mediumwithout fetal bovine serum during 48-72 hrs on 50 μW power of 488 nmwavelength blue light. After finishing culture, supernatant removed celldebris was yielded by centrifuge (2000×g, 15 min) from isolated culturemedium. To remove particles bigger than 200 nm from supernatant, itfiltered by 0.2 μl PES membrane (Corning). Tangential Flow Filtration(TFF) method was applied in identical supernatant to remove particlessmaller than 20 nm, condense and refine exosomes from filtrate. Vivaflow50-100 kDa PES membrane (Sartorius) was used in TFF. Exosomes werecondensed and refined by rotation of filtrate under 1.5˜2 air pressureof TFF. Then, exosome concentrate was eliminated liquid bycentrifugation (10000˜14000 g, 5 min) on Amicon Ultra-0.5 (100 kDa)(Millipore) filter. Finally, exosomes were obtained byreverse-directional centrifugation (10000˜14000 g, 5 min) withpreferable buffer on experimental purpose.

<5-2> Confirmation of Cre Recombinase Function by Cre Recombinase-LoadedExosome (Cre::EXPLOR)

A. Functional Confirmation of Cre::EXPLOR on HT1080 and HeLa Cell

Cre recombinase has the function to recombine DNA on loxP regions. Thepresent inventors performed following experiment to investigate thefunction of Cre::EXPLOR.

In particular, pCAG-loxP-STOP-loxP-ZsGreen encoded DNA was transfectedto HT1080 and HeLa cell and washed after 6 hrs. Then 0.25 mg/mlCre::EXPLOR or Negative::EXPLOR was treated or pCMV-Cre vector wastransfected. After 48 hrs culture, expression of ZsGreen with greenfluorescence was investigated. Expression of ZsGreen was confirmed onCre::EXPLOR treated HT1080 and HeLa cell, unlike Negative::EXPLORtreated HT1080 and HeLa cell, and was similar to the results of pCMV-Crevector transfection in positive control (FIGS. 19A and 19B).

B. Confirmation of Cre::EXPLOR's Function on Primary Rat EmbryonicNeuron

Following experiment was performed to investigate function ofCre::EXPLOR on primary rat embryonic neuron.

In particular, pCAG-loxP-STOP-loxP-ZsGreen encoded DNA was transfectedto primary rat embryonic neurons and washed after 6 hrs. Then they werecultured on 0.15 mg/ml Cre::EXPLOR. After 48 hrs culture, expression ofZsGreen with green fluorescence was investigated. This experiment wasperformed at least three repeats, and thereby expression of ZsGreen wasconfirmed on Cre::EXPLOR treated primary rat embryonic neuron (FIG. 20).

C. Confirmation of Cre::EXPLOR Function on In Vivo Transgenic Mouse

The present inventors were performed following experiment to verifyCre::EXPLOR's function on in vivo.

In particular, 50 μl Cre:EXPLORs (10 mg/mL) was injected byventrolateral injection to pCAG-loxP-STOP-loxP-eNpHR3.0-EYFP transgenicmouse. After injection, fixed brain slices by 4% formaldehyde wereimaged by fluorescence microscopy. Green fluorescence indicatesexpression of eNpHR3.0-EYFP, and blue fluorescence indicates cellnuclei. eNpHR3.0-EYFP expression on neuron in zona incerta (ZI) ofCre::EXPLOR treated mouse was investigated by confocal microscopy, andthereby EYFP expression was confirmed on Cre::EXPLOR treated groups ofpCAG-loxP-STOP-loxP-eNpHR3.0-EYFP transgenic mouse (FIG. 21).

D. Confirmation of Cre::EXPLOR Target Cell on Transgenic Mouse

To confirm specific cell targeting of Cre::EXPLOR on aforementioned invivo experiment, immunohistochemistry was performed. NeuN antibodyspecifically stained neuron, and GFAP antibody specifically stainedastrocytes, and thereby it was confirmed that Cre::EXPLOR targetsspecifically neuron in mouse brain through investigating that mergedregion mainly was neuron (FIG. 22).

Experimental Example 6: CRISPR-Cas9

<6-1> Production of Cas9-Loaded Exosome (Cas9::EXPLOR)

A. Confirmation of Cas9 within Exosome

The present inventors investigated the binding of CIBN and CRY2expressing the CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 to confirm theloading of Cas9, which is recorded in amino acid SEQ ID NO: 10.

As described in FIG. 23, Cas9-mCherry-CRY2 inserted pcDNA3.1(+) vectorhas 11,890 base pair in length, and the three protein parts consist ofCas9 with NLS sequence at 5-terminal, mCherry, and Cryptochrome 2 has 45and 27 base pairs of linker sequences, respectively. Each protein parthas 4194, 699, and 1497 base pairs in lengths, respectively.

In particular, HEK293T exosome producing cells were additionallycultured 48 hrs in Dulbecco's modified Eagle's medium (DMEM) withoutfetal bovine serum (FBS), after 24 hrs culture with transfectedpcDNA3.1(+) vector including CIBN-EGFP-CD9 gene and Cas9-mCherry-CRY2gene in non-light condition. After finish culture, position of redfluorescence from mCherry was investigated by confocal microscopy beforeand after the irradiation of 488 nm wavelength blue light. Thisexperiment was performed more than five times, and thereby Cas9 proteinis loaded within exosome by confirming that CIBN-EGFP-CD9 binds toCas9-mCherry-CRY2 by the blue light stimulus (FIG. 23).

B. Production of Cas9-Loaded Exosome (Cas9::EXPLOR)

The present inventors performed following experiment to yield theCas9-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and Cas9-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. After 24 hrs culture, cells were changedtheir medium as it without fetal bovine serum (FBS) and additionallycultured during 48 hrs on 50 μW power of 488 nm wavelength blue light.After finishing culture, supernatant removed cell debris was yielded bycentrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and Cas9-mCherry-CRY2 gene were cultured in mediumwithout fetal bovine serum during 48-72 hrs on 50 μW power of 488 nmwavelength blue light. After finishing culture, supernatant removed celldebris was yielded by centrifuge (2000×g, 15 min) from isolated culturemedium. To remove particles bigger than 200 nm from supernatant, itfiltered by 0.2 μl PES membrane (Corning). Tangential Flow Filtration(TFF) method was applied in identical supernatant to remove particlessmaller than 20 nm, condense and refine exosomes from filtrate. Vivaflow50-100 kDa PES membrane (Sartorius) was used in TFF. Exosomes werecondensed and refined by rotation of filtrate under 1.5˜2 air pressureof TFF. Then, exosome concentrate was eliminated liquid bycentrifugation (10000˜14000 g, 5 min) on Amicon Ultra-0.5 (100 kDa)(Millipore) filter. Finally, exosomes were obtained byreverse-directional centrifugation (10000˜14000 g, 5 min) withpreferable buffer on experimental purpose. The loading of Cas9 withinthe exosome was evaluated (FIG. 25).

Functional analysis of Cas9-loaded exosomes is performed in targetcells:

Target cells are treated with the Cas9-loaded exosomes to show thefunctional activity.

Animal models are administered with the Cas9-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 7: Caspase-Activated DNase (CAD)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andCAD-mCherry-Cry2 at 488 nm wavelength blue light, and the loading of CADwithin the exosome is evaluated.

For the massive production of CAD-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and CAD-mCherry-CRY2 gene are established,and exosomes are isolated and purified by Tangential Flow Filtration(TFF) method from culture supernatant.

Functional analysis of CAD-loaded exosomes is performed in target cells:

Target cells are treated with the CAD-loaded exosomes to show thefunctional activity.

Animal models are administered with the CAD-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 8: β-glucocerebrosidase

<8-1> Production of GBA-Loaded Exosome (GBA::EXPLOR)

A. Confirmation of GBA within Exosome

The present inventors investigated the binding of CIBN and CRY2expressing the CIBN-EGFP-CD9 and GBA-mCherry-CRY2 to confirm the loadingof GBA, which is recorded in amino acid SEQ ID NO: 12.

TABLE 2  SEQ. ID GENE Nucleotide sequence 12 β-gluco-MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAV cerebro-SWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPT sidaseFPALGTFSRYESTRSGRRMELSMGPIQANHTGTG LLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRT YTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQ PGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTL ANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPN TMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFV DSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSK DVPLTIKDPAVGFLETISPGYSIFITYLWRRQ

In particular, HEK293T exosome producing cells were additionallycultured 48 hrs in Dulbecco's modified Eagle's medium (DMEM) withoutfetal bovine serum (FBS), after 24 hrs culture with transfectedpcDNA3.1(+) vector including CIBN-EGFP-CD9 gene and GBA-mCherry-CRY2gene in non-light condition. After finish culture, position of redfluorescence from mCherry was investigated by confocal microscopy beforeand after the irradiation of 488 nm wavelength blue light. Thisexperiment was performed more than five times, and thereby GBA proteinis loaded within exosome by confirming that CIBN-EGFP-CD9 binds toGBA-mCherry-CRY2 by the blue light stimulus (FIG. 26).

B. Production of GBA-Loaded Exosome (GBA::EXPLOR)

The present inventors performed following experiment to yield theGBA-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. After 24 hrs culture, cells were changedtheir medium as it without fetal bovine serum (FBS) and additionallycultured during 48 hrs on 50 μW power of 488 nm wavelength blue light.After finishing culture, supernatant removed cell debris was yielded bycentrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and GBA-mCherry-CRY2 gene were cultured in mediumwithout fetal bovine serum during 48˜72 hrs on 50 μW power of 488 nmwavelength blue light. After finishing culture, supernatant removed celldebris was yielded by centrifuge (2000×g, 15 min) from isolated culturemedium. To remove particles bigger than 200 nm from supernatant, itfiltered by 0.2 μl PES membrane (Corning). Tangential Flow Filtration(TFF) method was applied in identical supernatant to remove particlessmaller than 20 nm, condense and refine exosomes from filtrate. Vivaflow50-100 kDa PES membrane (Sartorius) was used in TFF. Exosomes werecondensed and refined by rotation of filtrate under 1.5˜2 air pressureof TFF. Then, exosome concentrate was eliminated liquid bycentrifugation (10000˜14000 g, 5 min) on Amicon Ultra-0.5 (100 kDa)(Millipore) filter. Finally, exosomes were obtained byreverse-directional centrifugation (10000˜14000 g, 5 min) withpreferable buffer on experimental purpose.

<8-2> Measurement of GBA Expression in GBA-Loaded Exosome ProducingCells

The present inventors performed western blot to measure GBA expressionin GBA-loaded exosome.

In particular, CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. The HEK293T cells were lysed using MPER(Mammalian Protein Extraction Reagent) and the proteins were analyzed bywestern blot. Rat primary astrocyte, human primary astrocyte, andGaucher disease patient-derived fibroblast where β-glucocerebrosidase isdeficient due to GBA gene abnormality were lysed to perform western blotand the proteins were analyzed by western blot.

As a result, endogenous GBA was observed in HEK293T cells includingCIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene, rat primary astrocyte,human primary astrocyte, except Gaucher disease patient-derivedfibroblast (FIG. 27).

In addition, GBA-mCherry-CRY2 fusion protein (151 kDa) was observed inHEK293T cells including CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene,and this presents that GBA-mCherry-CRY2 fusion protein is well expressedin GBA-loaded exosome producing cells (FIG. 28).

<8-3> Confirmation of GBA Activity on Gaucher Disease Patient-DerivedCells by GBA-Loaded Exosome (GBA::EXPLOR)

A. Enzyme Activity of GBA within Exosome

The present inventor performed experiment for β-glucocerebrosidaseenzyme activity to investigate glucocerebroside degrading activity ofGBA within exosome.

In particular, CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. After 24 hrs culture, cells were changedtheir medium as it without fetal bovine serum (FBS) and additionallycultured during 48 hrs on 50 μW power of 488 nm wavelength blue light.After finishing culture, supernatant removed cell debris was yielded bycentrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture.

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and GBA-mCherry-CRY2 gene were cultured in mediumwithout fetal bovine serum during 48˜72 hrs on 50 μW power of 488 nmwavelength blue light. After finishing culture, supernatant removed celldebris was yielded by centrifuge (2000×g, 15 min) from isolated culturemedium. To remove particles bigger than 200 nm from supernatant, itfiltered by 0.2 μl PES membrane (Corning). Tangential Flow Filtration(TFF) method was applied in identical supernatant to remove particlessmaller than 20 nm, condense and refine exosomes from filtrate. Vivaflow50-100 kDa PES membrane (Sartorius) was used in TFF. Exosomes werecondensed and refined by rotation of filtrate under 1.5˜2 air pressureof TFF. Then, exosome concentrate was eliminated liquid bycentrifugation (10000˜14000 g, 5 min) on Amicon Ultra-0.5 (100 kDa)(Millipore) filter. Finally, exosomes were obtained byreverse-directional centrifugation (10000˜14000 g, 5 min). The exosomeswere lysed using MPER (Mammalian Protein Extraction Reagent) and theproteins were analyzed.

Increased β-glucocerebrosidase enzyme activity of GBA-loaded GBA::EXPLORwas observed comparing to mCherry-loaded exosomes, and thereby activeGBA loading on exosome was confirmed (FIG. 29).

B. Enzyme Activity of β-Glucocerebrosidase (GBA) on Gaucher DiseasePatients-Derived Cells

The present inventors performed following experiment to confirm therecovery of β-glucocerebrosidase enzyme activity on Gaucher diseasepatients-derived cells when treated with GBA::EXPLOR.

Gaucher disease patients-derived fibroblast was cultivated at thedensity of 2×10⁵ cells in 60 mm dish. Then, mCherry::EXPLORs (2×10⁹exosomes) or GBA::EXPLORs (1.2×10¹⁰ exosomes) were treated to Gaucherdisease patients-derived fibroblast cultured in serum-free DMEM medium.Hydrolysis activity of GBA-mCh-CRY2 was measured by detecting thefluorescence using substrate 4-methylumbelliferyl-β-D-glucopyranoside(4-MUG; Sigma). Enzyme reaction was performed on 0.2 ml of 0.2 M citratephosphate buffer (pH 0.5) containing 50 μl cell lysate of 0.15% (v/v)Triton X-100 (Sigma), 0.8% (w/v) sodium taurocholate (Sigma), 10 mM4-MUG. After 1 hr incubation at 37° C., the enzyme activity was stoppedusing 100 μl of 0.1 M glycine, 0.1 M NaOH (pH 10.3). Enzyme reactionproduct, 4-methylumbelliferone (4-MU) was measured at excitation 365 nm,emission 460 nm condition.

As a result, β-glucocerebrosidase enzyme activity of GBA-loadedGBA::EXPLOR treated Gaucher disease patients-derived cells was recovered(FIG. 30).

Experimental Example 9: Mitogen Activated Kinases: p38 MAP Kinase

The present inventors confirm the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and p38 MAP kinase-mCherry-Cry2 at 488 nmwavelength blue light, and verify the loading of p38 MAP kinase withinexosome.

For the massive production of p38 MAP kinase-loaded exosomes, cellsstably expressing CIBN-EGFP-CD9 gene and p38 MAP kinase-mCherry-CRY2gene are established, and exosomes are isolated and purified byTangential Flow Filtration (TFF) method from culture supernatant.

Functional analysis of p38 MAP kinase-loaded exosomes is performed intarget cells

Treatment of p38 MAP kinase-loaded exosomes to target cells shows thefunctional activity.

Administration of p38 MAP kinase-loaded exosomes by i.p. or i.v. toanimal model shows therapeutic effect.

Experimental Example 10: Inhibitor Kappa B Kinase (IKK)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andIKK-mCherry-Cry2 at 488 nm wavelength blue light, and the loading of IKKwithin the exosome is evaluated.

For the massive production of IKK-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and IKK-mCherry-CRY2 gene are established,and exosomes are isolated and purified by Tangential Flow Filtration(TFF) method from culture supernatant.

Functional analysis of IKK kinase-loaded exosomes is performed in targetcells:

Target cells are treated with the IKK-loaded exosomes to show thefunctional activity.

Animal models are administered with the IKK-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 11: PTEN Phosphatase

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and PTEN-Cry2 at 488 nm wavelength blue light,and the loading of PTEN within exosome.

For the massive production of PTEN-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and PTEN-CRY2 gene were established (FIG.31), and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of PTEN kinase-loaded exosomes is performed intarget cells

Treatment of PTEN-loaded exosomes to target cells shows the functionalactivity.

Administration of PTEN-loaded exosomes by i.p. or i.v. to animal modelshows therapeutic effect.

Experimental Example 12: Janus Kinase (JNK)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andJNK-mCherry-Cry2 at 488 nm wavelength blue light, and the loading of JNKwithin the exosome is evaluated.

For the massive production of JNK-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and JNK-mCherry-CRY2 gene are established,and exosomes are isolated and purified by Tangential Flow Filtration(TFF) method from culture supernatant.

Functional analysis of JNK-loaded exosomes is performed in target cells:

Target cells are treated with the JNK-loaded exosomes to show thefunctional activity.

Animal models are administered with the JNK-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 13: Ubiquitin Ligases

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andUbiquitin ligase-mCherry-Cry2 at 488 nm wavelength blue light, and theloading of Ubiquitin ligases within the exosome is evaluated.

For the massive production of Ubiquitin ligase-loaded exosomes, cellsstably expressing CIBN-EGFP-CD9 gene and Ubiquitin ligase-mCherry-CRY2gene are established, and exosomes are isolated and purified byTangential Flow Filtration (TFF) method from culture supernatant.

Functional analysis of Ubiquitin ligase-loaded exosomes is performed intarget cells:

Target cells are treated with the Ubiquitin ligase-loaded exosomes toshow the functional activity.

Animal models are administered with the Ubiquitin ligase-loaded exosomesby i.p. or i.v. to show therapeutic effect.

Experimental Example 14: Luciferase

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and luciferase-mcherry-Cry2 at 488 nmwavelength blue light, and the loading of luciferase within exosome(FIG. 32).

For the massive production of luciferase-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and luciferase-mcherry-CRY2 gene wereestablished, and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Quantitative luciferase activity based on the number of luciferasemolecules was analyzed (FIG. 33).

Experimental Example 15: Peroxiredoxin

<15-1> Production of Prx I or Prx II-Loaded Exosome (Prx I/II:EXPLOR)

A. Confirmation of Prx I/II within Exosome

The present inventors investigated the binding of CIBN and CRY2expressing the CIBN-EGFP-CD9 and Prx I/II-mCherry-CRY2 to confirm theloading of Prx I or Prx II, which is recorded in amino acid SEQ ID NO: 7or 8.

In particular, HEK293T exosome producing cells were additionallycultured 48 hrs in Dulbecco's modified Eagle's medium (DMEM) withoutfetal bovine serum (FBS), after 24 hrs culture with transfectedpcDNA3.1(+) vector including CIBN-EGFP-CD9 gene and PrxI/II-mCherry-CRY2 gene in non-light condition. After finish culture,position of red fluorescence from mCherry was investigated by confocalmicroscopy before and after the irradiation of 488 nm wavelength bluelight. This experiment was performed more than five times, and therebyaggregation of Prx I/II protein by blue light stimulation was confirmed(FIG. 34). Therefore, Prx I/II protein was loaded in exosome byconfirming the co-localization (yellow) of Prx I/II-mCherry-CRY2 (red)and CIBN-EGFP-CD9 (green).

B. Production of Prx I/II::EXPLOR

The present inventors performed following experiment to yield the PrxI/II-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and Prx I/II-mCherry-CRY2 geneincluded vectors were transfected on HEK293T exosome producing cells andthese cells were cultured 24 hrs. After 24 hrs culture, cells werechanged their medium as it without fetal bovine serum (FBS) andadditionally cultured during 48 hrs on 50 μW power of 488 nm wavelengthblue light. After finishing culture, supernatant removed cell debris wasyielded by centrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

<15-2> Confirmation of Inhibition Effect on Oxidative Stress-InducedCytotoxicity by Prx I/II::EXPLOR

The present inventors performed following experiment to confirm theinhibition effect on oxidative stress-induced cytotoxicity by PrxI/II::EXPLOR.

In particular, after changing the serum-free media of HeLa cells, 100μg/mL of Prx I/II::EXPLORs was treated and cultivated for 18 hrs. H₂O₂(0, 0.5, 1 mM) was treated and cultivated for additional 8 hrs. WSTassays were used to analyze the cell viability.

Due to pretreat of Prx I/II::EXPLORs, the oxidative stress-inducedcytotoxicity was significantly inhibited (FIG. 35).

Experimental Example 16: NF-κB

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andNF-κB-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofNF-κB within the exosome is evaluated.

For the massive production of NF-κB-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and NF-κB-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of NF-κB-loaded exosomes is performed in targetcells:

Target cells are treated with the NF-κB-loaded exosomes to show thefunctional activity.

Animal models are administered with the NF-κB-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 17: MyoD

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and MyoD-mcherry-Cry2 at 488 nm wavelength bluelight (FIG. 36), and verify the loading of MyoD within exosome.

For the massive production of MyoD-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and MyoD-mcherry-CRY2 gene wereestablished, and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Treatment of MyoD-loaded exosomes to target cells showed the functionalactivity (FIG. 37).

Experimental Example 18: Tbx18 (T-Box Transcription Factor 18)

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andTbx18-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofTbx18 within the exosome is evaluated.

For the massive production of Tbx18-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and Tbx18-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of Tbx18-loaded exosomes is performed in targetcells:

Target cells are treated with the Tbx18-loaded exosomes to show thefunctional activity.

Animal models are administered with the Tbx18-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 19: p53

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and p53-mcherry-Cry2 at 488 nm wavelength bluelight (FIG. 38), and the loading of PTEN within exosome (FIG. 39)

For the massive production of p53-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and p53-mcherry-CRY2 gene wereestablished, and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Treatment of p53-loaded exosomes to target cells showed thetranscriptional activity (FIG. 40).

Administration of p53-loaded exosomes by i.p. or i.v. to animal modelshows therapeutic effect.

Experimental Example 20: HMGB1

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and HMGB1-Cry2 at 488 nm wavelength blue light,and the loading of HMGB1 within exosome (FIG. 41).

For the massive production of HMGB1-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and HMGB1-CRY2 gene were established, andexosomes were isolated and purified by Tangential Flow Filtration (TFF)method from culture supernatant.

Functional analysis of HMGB1-loaded exosomes is performed in targetcells

Treatment of HMGB1-loaded exosomes to target cells shows thetranscriptional activity.

Administration of HMGB1-loaded exosomes by i.p. or i.v. to animal modelshows therapeutic effect.

Experimental Example 21: Super-Repressor IκB

<21-1> Production of Super-Repressor-IκB-Loaded Exosome(Super-Repressor-IκB:EXPLOR)

A. Confirmation of Super-Repressor-IκB in Exosome

The present inventors investigated the binding of CIBN and CRY2expressing the CIBN-EGFP-CD9 and super-repressor-IκB-mCherry-CRY2 toconfirm the loading of super-repressor-IκB, which is recorded in aminoacid SEQ ID NO: 5.

In particular, HEK293T exosome producing cells were additionallycultured 48 hrs in Dulbecco's modified Eagle's medium (DMEM) withoutfetal bovine serum (FBS), after 24 hrs culture with transfectedpcDNA3.1(+) vector including CIBN-EGFP-CD9 gene andsuper-repressor-IκB-mCherry-CRY2 gene in non-light condition. Afterfinish culture, position of red fluorescence from mCherry wasinvestigated by confocal microscopy before and after the irradiation of488 nm wavelength blue light. This experiment was performed more thanfive times, and thereby aggregation of super-repressor-IκB protein byblue light stimulation was confirmed (FIG. 42). Therefore,super-repressor-IκB protein was loaded in exosome by confirming theco-localization (yellow) of super-repressor-IκB-mCherry-CRY2 (red) andCIBN-EGFP-CD9 (green).

B. Production of Super-Repressor-IκB::EXPLOR

The present inventors performed following experiment to yield thesuper-repressor-IκB-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and super-repressor-IκB-mCherry-CRY2gene included vectors were transfected on HEK293T exosome producingcells and these cells were cultured 24 hrs. After 24 hrs culture, cellswere changed their medium as it without fetal bovine serum (FBS) andadditionally cultured during 48 hrs on 50 μW power of 488 nm wavelengthblue light. After finishing culture, supernatant removed cell debris wasyielded by centrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and super-repressor-IκB-mCherry-CRY2 gene werecultured in medium without fetal bovine serum during 48˜72 hrs on 50 μWpower of 488 nm wavelength blue light. After finishing culture,supernatant removed cell debris was yielded by centrifuge (2000×g, 15min) from isolated culture medium. To remove particles bigger than 200nm from supernatant, it filtered by 0.2 μl PES membrane (Corning).Tangential Flow Filtration (TFF) method was applied in identicalsupernatant to remove particles smaller than 20 nm, condense and refineexosomes from filtrate. Vivaflow 50-100 kDa PES membrane (Sartorius) wasused in TFF. Exosomes were condensed and refined by rotation of filtrateunder 1.5˜2 air pressure of TFF. Then, exosome concentrate waseliminated liquid by centrifugation (10000˜14000 g, 5 min) on AmiconUltra-0.5 (100 kDa) (Millipore) filter. Finally, exosomes were obtainedby reverse-directional centrifugation (10000˜14000 g, 5 min) withpreferable buffer on experimental purpose.

<21-2> Confirmation of Super-Repressor-IκB:EXPLOR Inhibition Effect ofTNF-α Mediated NF-κB Activity

The present inventors performed following experiments to confirm theTNF-α mediated anti-inflammatory effect usingsuper-repressor-IκB:EXPLOR.

In particular, HeLa was cultured in 100 mg/mL of mCherry:EXPLORs orsuper-repressor-IκB-mCherry:EXPLORs treated culture medium for 3 hrs.Then, TNF-α (10 ng/mL) was treated and incubated for additional 30minutes. After fixing with 4% paraformaldehyde, NF-κB p65 was stainedwith Alexa Fluor 488-conjugated antibody and inspected using confocalmicroscopy. To measure the binding activity of p65/c-Rel (NF-kB), nucleilysate was used in TransAM NF-kB and AP-1 assay kit (ActiveMotif,Carlsbad, Calif., USA) according to manufacturer's protocol. Data waspresented average±SEM (n=3), and applied using Tukey's post hoc test anddecided significant group (**, p<0.01) through ANOVA test.

By pretreat of super-repressor-IκB:EXPLOR on HeLa cells, TNF-α-activatedNF-κB transport to nucleus and NF-κB DNA binding were inhibited (FIG.43).

<21-3> Confirmation of Anti-Inflammatory Effect ofSuper-Repressor-IκB:EXPLOR on Collagen-Induced Arthritis Animal Model

The present inventors performed the following experiment to confirm theanti-inflammatory effect of super-repressor-IκB:EXPLOR onCollagen-induced arthritis mouse model.

In particular, mostly used rheumatoid arthritis model, collagen-inducedarthritis mouse model was developed by immunization through injectingbovine collagen type II and adjuvant to tail subcutaneous tissue ofDBA/1. Super repressor IκB:EXPLOR was retro-orbitally injected 4 timesto two collagen-induced arthritis mouse models every 2 days. Progressionof rheumatoid arthritis symptom was determined by clinical score aslisted in Table 3. Mean Clinical Score is average value of clinicalscores from mouse feet according to the aforementioned table.

When super repressor IκB:EXPLOR was retro-orbitally injected tocollagen-induced arthritis mouse models, the rheumatoid arthritis mouseshowed decreased symptom (FIG. 44).

TABLE 3 Severity Score Phenotypic signs 0 No evidence of erythema andswelling 1 Erythema and mild swelling confined to the tarsals or anklejoint 2 Erythema and mild swelling extending from the ankle to thetarsals 3 Erythema and moderate swelling extending from the ankle tometatarsal joint 4 Erythema and severe swelling encompass the ankle,foot and digits, or ankylosis of the limb

<21-4> Effect of srIkB-Loaded Exosomes on LPS-Induced Sepsis Model

In addition, when super repressor IκB:EXPLOR was Intraperitoneallyinjected to LPS-induced sepsis mouse models, the sepsis mouse showedsignificantly increased survival (FIG. 45).

Experimental Example 22: pySTAT3 Intrabody

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and pySTAT3-mcherry-Cry2 at 488 nm wavelengthblue light, and the loading of pySTAT3 within exosome (FIG. 46).

For the massive production of pySTAT3-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and pySTAT3-mcherry-CRY2 gene wereestablished, and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Treatment of pySTAT3-loaded exosomes to target cells shows thefunctional activity.

Administration of pySTAT3-loaded exosomes by i.p. or i.v. to animalmodel shows therapeutic effect.

Experimental Example 23: Bcl-2-Associated X Protein

<23-1> Production of Bax-Loaded Exosome (Bax::EXPLOR)

A. Confirmation of Bax in Exosome

The present inventors investigated the binding of CIBN and CRY2expressing the CIBN-EGFP-CD9 and Bax-mCherry-CRY2 to confirm the loadingof Bax, which is recorded in amino acid SEQ ID NO: 6.

In particular, HEK293T exosome producing cells were additionallycultured 48 hrs in Dulbecco's modified Eagle's medium (DMEM) withoutfetal bovine serum (FBS), after 24 hrs culture with transfectedpcDNA3.1(+) vector including CIBN-EGFP-CD9 gene and Bax-mCherry-CRY2gene in non-light condition. After finish culture, position of redfluorescence from mCherry was investigated by confocal microscopy beforeand after the irradiation of 488 nm wavelength blue light. Thisexperiment was performed more than five times, and thereby aggregationof Bax protein by blue light stimulation was confirmed (FIG. 48).Therefore, Bax protein was loaded in exosome by confirming the bindingof Bax-mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green).

B. Production of Bax::EXPLOR

The present inventors performed following experiment to yield theBax-loaded exosomes.

In particular, CIBN-EGRP-CD9 gene and Bax-mCherry-CRY2 gene includedvectors were transfected on HEK293T exosome producing cells and thesecells were cultured 24 hrs. After 24 hrs culture, cells were changedtheir medium as it without fetal bovine serum (FBS) and additionallycultured during 48 hrs on 50 μW power of 488 nm wavelength blue light.After finishing culture, supernatant removed cell debris was yielded bycentrifuge (2000×g, 15 min) from isolated culture medium. Thesupernatant was added ExoQuick-TC Exosome Precipitation Solution (SystemBiosciences, Mountain View, Calif., USA) with five times more volume andmixed during 18 hrs on 4° C. Suspended exosomes were obtained bysuspending exosome pellet through centrifugation (1500×g, 30 min) ofaforementioned supernatant and ExoQuick-TC mixture (FIG. 8).

In addition, HEK293T exosome producing cells which stably expressCIBN-EGFP-CD9 gene and Bax-mCherry-CRY2 gene were cultured in mediumwithout fetal bovine serum during 48˜72 hrs on 50 μW power of 488 nmwavelength blue light. After finishing culture, supernatant removed celldebris was yielded by centrifuge (2000×g, 15 min) from isolated culturemedium. To remove particles bigger than 200 nm from supernatant, itfiltered by 0.2 μl PES membrane (Corning). Tangential Flow Filtration(TFF) method was applied in identical supernatant to remove particlessmaller than 20 nm, condense and refine exosomes from filtrate. Vivaflow50-100 kDa PES membrane (Sartorius) was used in TFF. Exosomes werecondensed and refined by rotation of filtrate under 1.5˜2 air pressureof TFF. Then, exosome concentrate was eliminated liquid bycentrifugation (10000˜14000 g, 5 min) on Amicon Ultra-0.5 (100 kDa)(Millipore) filter. Finally, exosomes were obtained byreverse-directional centrifugation (10000˜14000 g, 5 min) withpreferable buffer on experimental purpose.

<23-2> Confirmation of Apoptosis by Bax:EXPLOR

Bax is apoptotic regulator, thus the BAX overexpression releasecytochrome c by binding to mitochondrial membrane, and inducing theapoptosis. The present inventors confirmed the excretion of cytochrome cusing Bax:EXPLOR.

In particular, HeLa in 0.1 mg/mL of mCherry:EXPLORs or Bax:EXPLORscontaining medium was cultured for 12 hrs. After fixing using 4%paraformaldehyde, to measure the excretion of cytochrome c, the HeLa wasstained with Alexa Fluor 647-conjugated antibody and imaged usingconfocal microscope and the ratio of cytochrome c was analyzed bycounting the number of cells (Scale bars, 20 μm). Data was presentedaverage±SEM (n=3), and applied using Tukey's post hoc test and decidedsignificant group (**, p<0.01) through ANOVA test.

As a result, larger amount of cytochrome c release was observed inBax:EXPLOR treated HeLa than mCherry:EXPLOR treated HeLa (FIG. 49).

Experimental Example 24: Bcl-xL

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andBcl-xL-mCherry-Cry2 at 488 nm wavelength blue light, and the loading ofBcl-xL within the exosome is evaluated.

For the massive production of Bcl-xL-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and Bcl-xL-mCherry-CRY2 gene areestablished, and exosomes are isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Functional analysis of Bcl-xL-loaded exosomes is performed in targetcells:

Target cells are treated with Bcl-xL-loaded exosomes to show thefunctional activity.

Animal models are administered with Bcl-xL-loaded exosomes by i.p. ori.v. to show therapeutic effect.

Experimental Example 25: AIMP

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and AIMP-mcherry-Cry2 at 488 nm wavelength bluelight (FIG. 50), and the loading of AIMP within exosome (FIG. 51).

For the massive production of AIMP-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and AIMP-mcherry-CRY2 gene wereestablished, and exosomes were isolated and purified by Tangential FlowFiltration (TFF) method from culture supernatant.

Treatment of AIMP-loaded exosomes to target cells shows the functionalactivity.

Administration of AIMP-loaded exosomes by i.p. or i.v. to animal modelshows therapeutic effect.

Experimental Example 26: mCherry (Fluorescent Protein)

The present inventors confirmed the binding of CIBN and CRY2 in cellsexpressing CIBN-EGFP-CD9 and mCherry-Cry2 at 488 nm wavelength bluelight (FIG. 52), and the loading of AIMP within exosome.

For the massive production of mCherry-loaded exosomes, cells stablyexpressing CIBN-EGFP-CD9 gene and mCherry-CRY2 gene were established,and exosomes were isolated and purified by Tangential Flow Filtration(TFF) method from culture supernatant.

Experimental Example 27: Nucleic Acid-Binding Proteins

The binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 andNucleic acid-binding protein-mCherry-Cry2 at 488 nm wavelength bluelight, and the loading of Nucleic acid-binding protein within theexosome is evaluated.

For the massive production of Nucleic acid-binding protein-loadedexosomes, cells stably expressing CIBN-EGFP-CD9 gene and Nucleicacid-binding protein-mCherry-CRY2 gene are established, and exosomes areisolated and purified by Tangential Flow Filtration (TFF) method fromculture supernatant.

Functional analysis of Nucleic acid-binding protein-loaded exosomes isperformed in target cells:

Target cells are treated with Nucleic acid-binding protein-loadedexosomes to show the functional activity.

Animal models are administered with Nucleic acid-binding protein-loadedexosomes by i.p. or i.v. to show therapeutic effect.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended Claims.

1-17. (canceled)
 18. A method for mass production of an exosomecontaining a cargo protein comprising: a) introducing a polynucleotideencoding a fusion protein composed of an exosome specific marker and acargo protein or a polynucleotide or a polynucleotide encoding a fusionprotein composed of an exosome specific marker and a firstphoto-specific binding protein (fusion protein I) and a polynucleotideencoding a fusion protein composed of a cargo protein and a secondphoto-specific binding protein that can be linked to the firstphoto-specific binding protein (fusion protein II) into the exosomeproduction cells; b) culturing the transformed cells of step a); and c)separating the cargo protein from the exosomal membrane to prepare anexosome containing a cargo protein.
 19. The method of claim 18, whereinthe transformed cells are prepared by introducing the polynucleotideencoding the fusion protein composed of the exosome specific marker andthe cargo protein.
 20. The method of claim 18, wherein the transformedcells are prepared by introducing the polynucleotide encoding the fusionprotein composed of the exosome specific marker and the firstphoto-specific binding protein (fusion protein I) and the polynucleotideencoding the fusion protein composed of the cargo protein and the secondphoto-specific binding protein that can be linked to the firstphoto-specific binding protein (fusion protein II).
 21. The method ofclaim 18, wherein the transformed cells are prepared by transfecting avector containing a polynucleotide encoding a fusion protein composed ofan exosome specific marker and a cargo protein or a first vectorcontaining a fusion protein composed of an exosome specific marker and afirst photo-specific binding protein (fusion protein I) and a secondvector containing a polynucleotide encoding a fusion protein composed ofa cargo protein and a second photo-specific binding protein (fusionprotein II) into the exosome production cells.
 22. The method of claim20, further comprising irradiating the cultured cells obtained from stepb) with light to cause the conjugation between the first photo-specificbinding protein and the second photo-specific binding protein.
 23. Themethod of claim 22, wherein the cargo protein is separated from theexosome membrane by terminating the irradiation after the production ofthe exosome.
 24. The method of claim 18, wherein the cargo protein issuper-repressor-IκB protein inhibiting NF-κB, Bax(Bcl-2-associated Xprotein), Peroxiredoxin I, Peroxiredoxin II, Cre recombinase, Cas9(CRISPR associated protein 9), Cpf1(CRISPR from Prevotella andFrancisella 1), or GBA(β-glucocerebrosidase).
 25. The method of claim18, wherein the exosome production cell is one or more cells selectedfrom the group consisting of B-lymphocytes, T-lymphocytes, dendriticcells, megakaryocytes, macrophages, stem cells, and tumor cells.
 26. Themethod of claim 18, wherein the exosome specific marker is CD9, CD63,CD81, or CD82.
 27. The method of claim 18, wherein the photo-specificbinding protein is one or more proteins selected from the groupconsisting of CIB (cryptochrome-interacting basichelix-loop-helixprotein), CIBN (N-terminal domain of CIB), PhyB (phytochrome B), PIF(phytochrome interacting factor), FKF1 (Flavinbinding, Kelch repeat,Fbox 1), GIGANTEA, CRY (chryptochrome), and PHR (phytolyase homologousregion).
 28. The method of claim 18, wherein when the firstphoto-specific binding protein is CIB or CIBN, the second photo-specificbinding protein is CRY or PHR, and the binding of the firstphoto-specific binding protein and the second photo-specific bindingprotein is achieved by the irradiation of the light with the wavelengthof 460˜490 nm.
 29. The method of claim 18, wherein when the firstphoto-specific binding protein is PhyB, the second photo-specificbinding protein is PIF, and the binding of the first photo-specificbinding protein and the second photo-specific binding protein isachieved by the irradiation of the light with the wavelength of 600˜650nm.
 30. The method of claim 18, wherein when the first photo-specificbinding protein is GIGANTEA, the second photo-specific binding proteinis FKF1, and the binding of the first photo-specific binding protein andthe second photo-specific binding protein is achieved by the irradiationof the light with the wavelength of 460˜490 nm.
 31. A vector for theproduction of an exosome comprising: (a) a first expression vectorcontaining a polynucleotide encoding a fusion protein of an exosomespecific marker and a first photo-specific binding protein (fusionprotein I); and (b) a second expression vector containing a multicloningsite and a polynucleotide encoding a second photo-specific bindingprotein to be linked to the first photo-specific binding protein above.32. The vector of claim 31, wherein the second expression vector is toexpress a fusion protein composed of the second photo-specific bindingprotein and a cargo protein (fusion protein II).
 33. An exosomecontaining a cargo protein prepared by the method of claim
 18. 34. Amethod for delivering a protein drug to cytosol comprising administeringthe exosome of claim
 33. 35. A method for treating a medical disordercomprising: administering a pharmaceutically effective dose of theexosome containing the cargo protein of claim 33 as an activeingredient, wherein the medical disorder is Gaucher's disease, reactiveoxygen-related diseases, inflammatory diseases or cancer; and the cargoprotein is selected from β-glucocerebrosidase. Peroxiredoxin I orPeroxiredoxin II protein, Super-repressor IκB protein, Super-repressorIκB protein or Bax protein.